U.S. patent number 7,851,437 [Application Number 12/621,406] was granted by the patent office on 2010-12-14 for drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease.
This patent grant is currently assigned to Seattle Genetics Inc.. Invention is credited to Svetlana O. Doronina, Peter D. Senter, Brian E. Toki.
United States Patent |
7,851,437 |
Senter , et al. |
December 14, 2010 |
Drug conjugates and their use for treating cancer, an autoimmune
disease or an infectious disease
Abstract
Drug-Linker-Ligand Conjugates are disclosed in which a Drug is
linked to a Ligand via a peptide-based Linker unit. In one
embodiment, the Ligand is an Antibody. Drug-Linker compounds and
Drug compounds are also disclosed. Methods for treating cancer, an
autoimmune disease or an infectious disease using the compounds and
compositions of the invention are also disclosed.
Inventors: |
Senter; Peter D. (Seattle,
WA), Doronina; Svetlana O. (Snohomish, WA), Toki; Brian
E. (Shoreline, WA) |
Assignee: |
Seattle Genetics Inc. (Bothell,
WA)
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Family
ID: |
31188683 |
Appl.
No.: |
12/621,406 |
Filed: |
November 18, 2009 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20100062008 A1 |
Mar 11, 2010 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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12408646 |
Mar 20, 2009 |
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10522911 |
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7659241 |
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PCT/US03/24209 |
Jul 31, 2003 |
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60400403 |
Jul 31, 2002 |
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Current U.S.
Class: |
424/178.1;
514/19.3 |
Current CPC
Class: |
A61K
47/6867 (20170801); A61K 47/642 (20170801); A61K
47/6817 (20170801); A61K 47/64 (20170801); A61P
37/00 (20180101); A61P 31/00 (20180101); C07K
5/0205 (20130101); A61K 47/6851 (20170801); A61P
35/00 (20180101); A61P 37/06 (20180101) |
Current International
Class: |
A61K
38/00 (20060101) |
References Cited
[Referenced By]
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WO 2007/109567 |
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Primary Examiner: Bradley; Christina
Attorney, Agent or Firm: Townsend and Townsend and Crew
LLP
Parent Case Text
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser.
No. 12/408,646, filed Mar. 20, 2009, which is a continuation of
U.S. patent application Ser. No. 10/522,911, filed Jul. 7, 2005,
which was filed under 35 U.S.C. .sctn.371 as a national stage
application of International Application No. PCT/US2003/24209,
filed Jul. 31, 2003; which further claims the benefit under 35
U.S.C. .sctn.119(e) of U.S. Provisional Patent Application No.
60/400,403, filed Jul. 31, 2002. This application is also a
continuation of U.S. patent application Ser. No. 10/522,911, filed
Jul. 7, 2005, which was filed under 35 U.S.C. .sctn.371 as a
national stage application of International Application No.
PCT/US2003/24209, filed Jul. 31, 2003; which further claims the
benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional Patent
Application No. 60/400,403, filed Jul. 31, 2002. The disclosures of
each of the foregoing applications are hereby incorporated herein
by reference.
Claims
What is claimed is:
1. A drug-linker-antibody conjugate of Formula Ia: ##STR00215## or
a pharmaceutically acceptable salt thereof, wherein, L- is an
antibody that binds to an antigen expressed on an activated human
lymphocyte, wherein the activated human lymphocyte is associated
with an autoimmune disease; -A.sub.a-W.sub.w-Y.sub.y- is an
enzymatically cleavable linker unit that links the Drug unit and
the antibody, wherein: -A- is a Stretcher unit; a is 1; each -W- is
independently an Amino Acid unit; -Y- is a self-immolative Spacer
unit; w is an integer ranging from 2 to 12, y is 1 or 2; p ranges
from 1 to about 20; and -D is a Drug unit of the formula
##STR00216## wherein, the wavy line indicates the point of
attachment to the Spacer unit, and for each D: R.sup.2 is selected
from the group consisting of --H and --C.sub.1-C.sub.8 alkyl;
R.sup.3 is selected from the group consisting of --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from the
group consisting of --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from the group consisting of --H and -methyl; or R.sup.4 and
R.sup.5 join and form a ring with the carbon atom to which they are
attached and R.sup.4 and R.sup.5 have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from the group consisting of --H,
--C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8 carbocycle and n is
selected from the group consisting of 2, 3, 4, 5 and 6; R.sup.6 is
selected from the group consisting of --H and --C.sub.1-C.sub.8
alkyl; R.sup.7 is selected from the group consisting of --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from the group consisting of --H, --OH, --C.sub.1-C.sub.8
alkyl, --C.sub.3-C.sub.8 carbocycle and --O--(C.sub.1-C.sub.8
alkyl); R.sup.9 is selected from the group consisting of --H and
--C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from the group
consisting of: ##STR00217## Z is --O--, --S--,--NH--or
--N(R.sup.14)--; R.sup.11 is selected from the group consisting of
--H, --OH, --NH.sub.2, --NHR.sup.14, --N(R.sup.14).sub.2,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from the group consisting of -aryl and
--C.sub.3-C.sub.8 heterocycle; R.sup.13 is selected from the group
consisting of --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-8
alkyl-(C.sub.3-C.sub.8 heterocycle); and each R.sup.14 is
independently --H or --C.sub.1-C.sub.8 alkyl.
2. A drug-linker-antibody conjugate of the formula Ia: ##STR00218##
or a pharmaceutically acceptable salt thereof, wherein, L- is an
antibody that binds to an antigen expressed on an activated human
lymphocyte, wherein the activated human lymphocyte is associated
with an autoimmune disease; -A.sub.a-W.sub.w-Y.sub.y- is an
enzymatically cleavable linker unit that links the Drug unit and
the antibody, wherein: -A- is a Stretcher unit; a is 1; each -W- is
independently an Amino Acid unit; -Y- is a self-immolative Spacer
unit; w is an integer ranging from 2 to 12; y is 1 or 2; p ranges
from 1 to about 20; and -D is a Drug unit having the structure
##STR00219## wherein, the wavy line indicates the point of
attachment to the Spacer unit, and for each D: R.sup.2 is selected
from the group consisting of --H and -methyl; R.sup.3 is selected
from the group consisting of --H, -methyl, and -isopropyl; R.sup.4
is selected from the group consisting of --H and -methyl; R.sup.5
is selected from the group consisting of -isopropyl, -isobutyl,
-sec-butyl, -methyl and -t-butyl or R.sup.4 and R.sup.5 join and
form a ring with the carbon atom to which they are attached and
R.sup.4 and R.sup.5 have the formula --(CR.sup.aR.sup.b).sub.n--
where R.sup.a and R.sup.b are independently selected from the group
consisting of --H, --C.sub.1-C.sub.8 alkyl, and --C.sub.3-C.sub.8
carbocycle, and n is selected from the group consisting of 2, 3, 4,
5 and 6; R.sup.6 is selected from the group consisting of --H and
-methyl; each R.sup.8 is independently selected from the group
consisting of --OH, -methoxy and -ethoxy; R.sup.10 is selected from
the group consisting of: ##STR00220## R.sup.24 is selected from the
group consisting of H and --C(O)R.sup.25; wherein R.sup.25 is
selected from the group consisting of --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); Z is --O--, --NH--, --OC(O)--,
--NHC(O)--, or --NR.sup.28C(O)--; where R.sup.28 is selected from
the group consisting of --H and --C.sub.1-C.sub.8 alkyl; n is 0 or
1; and R.sup.27 is selected from the group consisting of --H,
--N.sub.3, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
-aryl, --C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1- C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle) when n
is 0; and R.sup.27 is selected from the group consisting of --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle, -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle) when n is
1.
3. The drug-linker-antibody conjugate of claim 1 or a
pharmaceutically acceptable salt thereof wherein R.sup.10 is
##STR00221##
4. The drug-linker-antibody conjugate of claim 2 or a
pharmaceutically acceptable salt thereof wherein R.sup.10 is
##STR00222##
5. The drug-linker-antibody conjugate of claim 3 or a
pharmaceutically acceptable salt thereof wherein R.sup.2 is
--C.sub.1-C.sub.8 alkyl.
6. The drug-linker-antibody conjugate of claim 4 or a
pharmaceutically acceptable salt thereof wherein R.sup.2 is
methyl.
7. The drug-linker-antibody conjugate of claim 1 or a
pharmaceutically acceptable salt thereof where -D is a Drug unit
having the structure ##STR00223##
8. The drug-linker-antibody conjugate of claim 1 or a
pharmaceutically acceptable salt thereof wherein the antibody is a
monoclonal antibody.
9. The drug-linker-antibody conjugate of claim 1 or a
pharmaceutically acceptable salt thereof wherein the antibody is a
full length immunoglobulin molecule.
10. The drug-linker-antibody conjugate of claim 8 or a
pharmaceutically acceptable salt thereof wherein the antibody
comprises a human immunoglobulin constant region.
11. The drug-linker-antibody conjugate of claim 10 or a
pharmaceutically acceptable salt thereof wherein the antibody is an
IgG 1.
12. The drug-linker-antibody conjugate of claim 1 or a
pharmaceutically acceptable salt thereof where -W.sub.w- is
-valine-citrulline-, the amino terminus of -W.sub.w- forming a bond
with a Stretcher unit, and the C-- terminus of -W.sub.w- forming a
bond with a Spacer unit.
13. The drug-linker-antibody conjugate of claim 1 or a
pharmaceutically acceptable salt thereof wherein p ranges from 1 to
about 5.
14. The drug-linker-antibody conjugate of claim 1 or a
pharmaceutically acceptable salt thereof wherein p ranges from 1 to
10.
15. The drug-linker-antibody conjugate of claim 1 or a
pharmaceutically acceptable salt thereof wherein the linker unit is
cleavable by cathepsin B.
16. The drug-linker-antibody conjugate of claim 1 having the
formula below: ##STR00224## or a pharmaceutically acceptable salt
thereof, where E is --CH.sub.2-- or --CH.sub.2CH.sub.2O--; e is an
integer ranging either from 0-10 when E is --CH.sub.2--, or from
1-10 when E is --CH.sub.2CH.sub.2--O--; F is --CH.sub.2--; f is 0
or 1; and p ranges from 1 to about 20.
17. The drug-linker-antibody conjugate of claim 16 or a
pharmaceutically acceptable salt thereof wherein the antibody is a
monoclonal antibody.
18. The drug-linker-antibody conjugate of claim 16 or a
pharmaceutically acceptable salt thereof wherein the antibody is a
full length immunoglobulin molecule.
19. The drug-linker-antibody conjugate of claim 17 or a
pharmaceutically acceptable salt thereof wherein the antibody
comprises a human immunoglobulin constant region.
20. The drug-linker-antibody conjugate of claim 19 or a
pharmaceutically acceptable salt thereof wherein the antibody is an
IgG1.
21. The drug-linker-antibody conjugate of claim 16 or a
pharmaceutically acceptable salt thereof wherein p ranges from 1 to
about 5.
22. The drug-linker-antibody conjugate of claim 16 or a
pharmaceutically acceptable salt thereof wherein p ranges from 1 to
10.
23. The drug-linker-antibody conjugate of claim 1 having the
formula below: ##STR00225## or a pharmaceutically acceptable salt
thereof.
24. The drug-linker-antibody conjugate of claim 23 or a
pharmaceutically acceptable salt thereof wherein the antibody is a
monoclonal antibody.
25. The drug-linker-antibody conjugate of claim 23 or a
pharmaceutically acceptable salt thereof wherein the antibody is
full length immunoglobulin molecule.
26. A composition comprising drug-linker-antibody conjugates having
Formula Ia: ##STR00226## or a pharmaceutically acceptable salt
thereof; wherein, L- is an antibody that binds to an antigen
expressed on an activated human lymphocyte, wherein the activated
human lymphocyte is associated with an autoimmune disease;
-A.sub.a-W.sub.w-Y.sub.y- is an enzymatically cleavable linker unit
that links the Drug unit and the antibody, wherein: -A- is a
Stretcher unit; a is 1; each -W- is independently an Amino Acid
unit; -Y- is a self-immolative Spacer unit; w is an integer ranging
from 2 to 12; y is 1 or 2; p ranges from 1 to 10 and is the average
number of -A.sub.a-W.sub.w-Y.sub.y-D units per antibody in the
composition; and -D is a Drug unit of the formula ##STR00227##
wherein, the wavy line indicates the point of attachment to the
Spacer unit, and for each D: R.sup.2 is selected from the group
consisting of --H and --C.sub.1-C.sub.8 alkyl; R.sup.3 is selected
from the group consisting of --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from the group consisting of --H, --C.sub.1-C.sub.8
alkyl, --C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl),
-aryl, --C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle) wherein
R.sup.5 is selected from the group consisting of --H and -methyl;
or R.sup.4 and R.sup.5 join and form a ring with the carbon atom to
which they are attached and R.sup.4 and R.sup.5 have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from the group consisting of --H,
--C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8 carbocycle and n is
selected from the group consisting of 2, 3, 4, 5 and 6; R.sup.6 is
selected from the group consisting of --H and --C.sub.1-C.sub.8
alkyl; R.sup.7 is selected from the group consisting of --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from the group consisting of --H, --OH, --C.sub.1-C.sub.8
alkyl, --C.sub.3-C.sub.8 carbocycle and --O--(C.sub.1-C.sub.8
alkyl); R.sup.9 is selected from the group consisting of --H and
--C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from the group
consisting of: ##STR00228## Z is --O--, --S--,--NH-- or
--N(R.sup.14)--; R.sup.11 is selected from the group consisting of
--H, --OH, --NH.sub.2, --NHR.sup.14, --N(R.sup.14).sub.2,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from the group consisting of -aryl and
--C.sub.3-C.sub.8 heterocycle; R.sup.13 is selected from the group
consisting of --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
C.sub.3-C.sub.8 heterocycle and --C.sub.1-8 alkyl-(C.sub.3-C.sub.8
heterocycle); and each R.sup.14 is independently --H or
--C.sub.1-C.sub.8 alkyl; and a pharmaceutically acceptable carrier
or vehicle.
27. A composition comprising drug-linker-antibody conjugates having
Formula Ia: ##STR00229## or a pharmaceutically acceptable salt
thereof, wherein, L- is an antibody that binds to an antigen
expressed on an activated human lymphocyte, wherein the activated
human lymphocyte is associated with an autoimmune disease;
-A.sub.a-W.sub.w-Y.sub.y- is an enzymatically cleavable linker unit
that links the Drug unit and the antibody , wherein: -A- is a
Stretcher unit; a is 1; each -W- is independently an Amino Acid
unit; -Y- is a self-immolative Spacer unit; w is an integer ranging
from 2 to 12; y is 1 or 2; p ranges from 1 to 10 and is the average
number of -A.sub.a-W.sub.w-Y.sub.y-D units per antibody in the
composition; and -D is a Drug unit having the structure
##STR00230## wherein, the wavy line indicates the point of
attachment to the Spacer unit, and for each D: R.sup.2 is selected
from the group consisting of --H and -methyl; R.sup.3 is selected
from the group consisting of --H, -methyl, and -isopropyl; R.sup.4
is selected from the group consisting of --H and -methyl; R.sup.5
is selected from the group consisting of -isopropyl, -isobutyl,
-sec-butyl, -methyl and -t-butyl or R.sup.4 and R.sup.5 join and
form a ring with the carbon atom to which they are attached and
R.sup.4 and R.sup.5 have the formula --(CR.sup.aR.sup.b).sub.n--
where R.sup.a and R.sup.b are independently selected from the group
consisting of --H, --C.sub.1-C.sub.8 alkyl, and --C.sub.3-C.sub.8
carbocycle, and n is selected from the group consisting of 2, 3, 4,
5 and 6; R.sup.6 is selected from the group consisting of --H and
-methyl; each R.sup.8 is independently selected from the group
consisting of --OH, -methoxy and -ethoxy; R.sup.10 is selected from
the group consisting of: ##STR00231## R.sup.24 is selected from the
group consisting of H and --C(O)R.sup.25--; wherein R.sup.25 is
selected from the group consisting of --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1 -C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); Z is --O--, --NH--, --OC(O)--,
--NHC(O)--, or --NR.sup.28C(O)--; where R.sup.28 is selected from
the group consisting of --H and --C.sub.1-C.sub.8 alkyl; n is 0 or
1; and R.sup.27 is selected from the group consisting of --H,
--N.sub.3, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
-aryl, --C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8alkyl-(C.sub.3-C.sub.8 heterocycle) when n is
0; and R.sup.27 is selected from the group consisting of --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle, -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-( C.sub.3-C.sub.8 heterocycle) when n
is 1; and a pharmaceutically acceptable carrier or vehicle.
28. The composition of claim 26 wherein in the drug-linker-antibody
conjugates or pharmaceutically acceptable salt thereof, R.sup.2 is
--C.sub.1-C.sub.8 alkyl.
29. The composition of claim 27 wherein in the drug-linker-antibody
conjugates or pharmaceutically acceptable salt thereof, R.sup.2 is
methyl.
30. The composition of claim 26 wherein in the drug-linker-antibody
conjugates or pharmaceutically acceptable salt thereof, -D is a
Drug unit having the structure ##STR00232##
31. The composition of claim 26 wherein the drug-linker-antibody
conjugates have the formula ##STR00233## or a pharmaceutically
acceptable salt thereof, where E is --CH.sub.2-- or
--CH.sub.2CH.sub.2O--; e is an integer ranging either from 0-10
when E is --CH.sub.2--, or from 1-10 when E is
--CH.sub.2CH.sub.2--O--; F is --CH.sub.2--; and f is 0 or 1.
32. The composition of claim 31 wherein in the drug-linker-antibody
conjugates or pharmaceutically acceptable salt thereof, the
antibody is a monoclonal antibody.
33. The composition of claim 31 wherein in the drug-linker-antibody
conjugates or pharmaceutically acceptable salt thereof, the
antibody is a full length immunoglobulin molecule.
34. The drug-linker-antibody conjugate of claim 32 or a
pharmaceutically acceptable salt thereof wherein the antibody
comprises a human immunoglobulin constant region.
35. The drug-linker-antibody conjugate of claim 34 or a
pharmaceutically acceptable salt thereof wherein the antibody is an
IgG 1.
36. The composition of claim 31 wherein in the drug-linker-antibody
conjugates or pharmaceutically acceptable salt thereof, p ranges
from 1 to about 5.
37. The composition of claim 26 wherein the drug-linker-antibody
conjugates have the formula ##STR00234## or a pharmaceutically
acceptable salt thereof.
38. The composition of claim 37 wherein in the drug-linker-antibody
conjugates or pharmaceutically acceptable salt thereof, the
antibody is a monoclonal antibody.
39. The composition of claim 37 wherein in the drug-linker-antibody
conjugates or pharmaceutically acceptable salt thereof, the
antibody is a full length immunoglobulin molecule.
40. The drug-linker-antibody conjugate of claim 38 or a
pharmaceutically acceptable salt thereof wherein the antibody
comprises a human immunoglobulin constant region.
41. The drug-linker-antibody conjugate of claim 40 or a
pharmaceutically acceptable salt thereof wherein the antibody is an
IgG1.
42. The composition of claim 37 wherein in the drug-linker-antibody
conjugates or pharmaceutically acceptable salt thereof, p ranges
from 1 to about 5.
Description
FIELD OF THE INVENTION
The present invention is directed to Drug-Linker-Ligand Conjugates
and to Drug-Linker Compounds, to compositions comprising a
Drug-Linker-Ligand Conjugate or a Drug-Linker Compound, and to
methods for using the same to treat cancer, an autoimmune disease
or an infectious disease.
BACKGROUND OF THE INVENTION
Several short peptidic compounds have been isolated from natural
sources and found to have biological activity. Analogs of these
compounds have also been prepared, and some were found to have
biological activity. For example, Auristatin E (U.S. Pat. No.
5,635,483 to Pettit et al.) is a synthetic analogue of the marine
natural product Dolastatin 10, an agent that inhibits tubulin
polymerization by binding to the same site on tubulin as the
anticancer drug vincristine (G. R. Pettit, Prog. Chem. Org. Nat.
Prod., 70: 1-79 (1997)). Dolastatin 10, auristatin PE, and
auristatin E are linear peptides having four amino acids, three of
which are unique to the dolastatin class of compounds. Both
dolastatin 10 and auristatin PE are presently being used in human
clinical trials to treat cancer. The structural differences between
dolastatin 10 and auristatin E reside in the C-terminal residue, in
which the thiazolephenethyl amine group of dolastatin 10 is
replaced by a norephedrine unit in auristatin E.
The following references disclose dolastatin and auristatin
compounds and analogs thereof, and their use for treating
cancer:
International Publication No. WO 96/33212 A1 to Teikoku Hormone
Mfg. Co., Ltd.;
International Publication No. WO 96/14856 A1 to Arizona Board of
Regents;
European Patent Publication No. EP 695757 A2 to Arizona Board of
Regents;
European Patent Publication No. EP 695758 A2 to Arizona Board of
Regents;
European Patent Publication No. EP 695759 A2 to Arizona Board of
Regents;
International Publication No. WO 95/09864 A1 to Teikoku Hormone
Mfg. Co., Ltd.;
International Publication No. WO 93/03054 A1 to Teikoku Hormone
Mfg. Co., Ltd.;
U.S. Pat. No. 6,323,315 B1 to Pettit et al.;
G. R. Pettit et al., Anti-Cancer Drug Des. 13(4): 243-277
(1998);
G. R. Pettit et al., Anti-Cancer Drug Des. 10(7): 529-544 (1995);
and
K. Miyazaki et al., Chem. Pharm. Bull. 43(10), 1706-18 (1995).
Despite in vitro data for compounds of the dolastatin class and its
analogs, significant general toxicities at doses required for
achieving a therapeutic effect compromise their efficacy in
clinical studies. Accordingly, there is a clear need in the art for
dolastatin derivatives having significantly lower toxicity, yet
useful therapeutic efficiency, compared to current dolastatin drug
therapies.
The recitation of any reference in Section 2 of this application is
not an admission that the reference is prior art to this
application.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides compounds of general
Formula Ia:
##STR00001## and pharmaceutically acceptable salts and solvates
thereof wherein, L- is a Ligand unit; -A- is a Stretcher unit; a is
0 or 1; each -W- is independently an Amino Acid unit; -Y- is a
Spacer unit; w is an integer ranging from 0 to 12; y is 0, 1 or 2;
p ranges from 1 to about 20; and -D is a Drug unit of the
formula
##STR00002## wherein, independently at each location: R.sup.2 is
selected from -hydrogen and --C.sub.1-C.sub.8 alkyl; R.sup.3 is
selected from -hydrogen, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from
-hydrogen, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from
##STR00003## Z is --O--, --S--, --NH-- or --N(R.sup.14)--; R.sup.11
is selected from --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; R.sup.13 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); and each
R.sup.14 is independently --H or --C.sub.1-C.sub.8 alkyl.
In another aspect, the present invention provides compounds of
general formula Ib:
##STR00004## and pharmaceutically acceptable salts and solvates
thereof wherein, L- is a Ligand unit; -A- is a Stretcher unit; a is
0 or 1; each -W- is independently an Amino Acid unit; -Y- is a
Spacer unit; w is an integer ranging from 0 to 12; y is 0, 1 or 2;
p ranges from 1 to about 20; and -D is a Drug unit of the
formula
##STR00005## wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from
##STR00006## X is --O--, --S--, --NH-- or --N(R.sup.14)--, where X
is bonded to Y when y is 1 or 2, or X is bonded to W when y is 0; Z
is --O--, --S--, --NH-- or --N(R.sup.14)--; R.sup.11 is selected
from --H, --OH, --NH.sub.2, --NHR.sup.14, --N(R.sup.14).sub.2,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; R.sup.13 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); each
R.sup.14 is independently --H or --C.sub.1-C.sub.8 alkyl; and
R.sup.15 is -arylene-, --C.sub.3-C.sub.8 carbocyclo- or
--C.sub.3-C.sub.8 heterocyclo-.
In another aspect, the present invention provides compounds of
general formula Ic:
##STR00007## and pharmaceutically acceptable salts and solvates
thereof wherein, L- is a Ligand unit; -A- is a Stretcher unit; a is
0 or 1; each -W- is independently an Amino Acid unit; w is an
integer ranging from 0 to 12; each n is independently 0 or 1; p
ranges from 1 to about 20; and each -D is independently: (a) a Drug
unit of the formula:
##STR00008## wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, -C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from
##STR00009## X is --O--, --S--, --NH-- or --N(R.sup.14)--, where X
is bonded to --C(O)-- when y is 1 or 2, or X is bonded to
--CH.sub.2-- when n is 0; Z is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; R.sup.13 is selected from --H, --OH,
--NH.sub.2, --NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8
alkyl, --C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl),
-aryl, --C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); each
R.sup.14 is independently --H or --C.sub.1-C.sub.8 alkyl; and
R.sup.15 is -arylene-, --C.sub.3-C.sub.8 carbocyclo- or
--C.sub.3-C.sub.8 heterocyclo-; or (b) a Drug unit of the
formula:
##STR00010## wherein, independently at each location: R.sup.2 is
selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.3 is selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from
##STR00011## Z is --O--, --S--, --NH-- or --N(R.sup.14)--; R.sup.11
is selected from --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; R.sup.13 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); and each
R.sup.14 is independently --H or --C.sub.1-C.sub.8 alkyl.
A compound of formula Ia, formula Ib, formula Ic or a
pharmaceutically acceptable salt or solvate thereof (a
"Drug-Linker-Ligand Conjugate") is useful for treating or
preventing cancer, an autoimmune disease or an infectious disease
in an animal.
In another aspect, the present invention provides compounds of the
formula IIa:
##STR00012## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; each R.sup.14 is independently --H
or --C.sub.1-C.sub.8 alkyl; and R.sup.16 is -Yy-Ww-A' wherein each
-W- is independently an Amino Acid unit; -Y- is a Spacer unit; w is
an integer ranging from 0 to 12; y is 0, 1 or 2; and -A' is
selected from
##STR00013## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O--(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IIb:
##STR00014## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; R.sup.13 is selected from hydrogen, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
alkyl-aryl, alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.14
is independently --H or --C.sub.1-C.sub.8 alkyl; R.sup.15 is
-arylene-, --C.sub.3-C.sub.8 carbocyclo- or --C.sub.3-C.sub.8
heterocyclo-; and R.sup.16 is -Yy-Ww-A' wherein each -W- is
independently an Amino Acid unit; -Y- is a Spacer unit; w is an
integer ranging from 0 to 12; y is 0, 1 or 2; and -A' is selected
from
##STR00015## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IIc:
##STR00016## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; each R.sup.14 is independently --H
or --C.sub.1-C.sub.8 alkyl; R.sup.16 is -Yy-Ww-A' wherein each -W-
is independently an Amino Acid unit; -Y- is a Spacer unit; w is an
integer ranging from 0 to 12; y is 0, 1 or 2; and -A' is selected
from
##STR00017## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IId:
##STR00018## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; each R.sup.14 is independently --H
or --C.sub.1-C.sub.8 alkyl; R.sup.15 is -arylene-,
--C.sub.3-C.sub.8 carbocyclo- or --C.sub.3-C.sub.8 heterocyclo-;
R.sup.16 is -Yy-Ww-A' wherein each -W- is independently an Amino
Acid unit; -Y- is a Spacer unit; w is an integer ranging from 0 to
12; y is 0, 1 or 2; and -A' is selected from
##STR00019## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl
In another aspect, the present invention provides compounds of the
formula IIe:
##STR00020## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--; Z is --O--, --S--, --NH-- or --N(R.sup.14)--;
R.sup.11 is selected from --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; each R.sup.14 is independently --H or
--C.sub.1-C.sub.8 alkyl; R.sup.15 is -arylene-, --C.sub.3-C.sub.8
carbocyclo- or --C.sub.3-C.sub.8 heterocyclo-; R.sup.16 is
-Yy-Ww-A' wherein each -W- is independently an Amino Acid unit; -Y-
is a Spacer unit; w is an integer ranging from 0 to 12; y is 0, 1
or 2; and -A' is selected from
##STR00021## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IIf:
##STR00022## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--; Z is --O--, --S--, --NH-- or --N(R.sup.14)--;
R.sup.11 is selected from --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; each R.sup.14 is independently --H or
--C.sub.1-C.sub.8 alkyl; R.sup.15 is -arylene-, --C.sub.3-C.sub.8
carbocyclo- or --C.sub.3-C.sub.8 heterocyclo-; R.sup.16 is
-Yy-Ww-A' wherein each -W- is independently an Amino Acid unit; -Y-
is a Spacer unit; w is an integer ranging from 0 to 12; y is 0, 1
or 2; and -A' is selected from
##STR00023## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IIg:
##STR00024## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.2 is
selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.3 is selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; Z is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; each R.sup.14 is independently --H
or --C.sub.1-C.sub.8 alkyl; R.sup.16 is -Yy-Ww-A' wherein each -W-
is independently an Amino Acid unit; -Y- is a Spacer unit; w is an
integer ranging from 0 to 12; y is 0, 1 or 2; and -A' is selected
from
##STR00025## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IIh:
##STR00026## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.2 is
selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.3 is selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; Z is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; each R.sup.14 is independently --H
or --C.sub.1-C.sub.8 alkyl; R.sup.16 is -Yy-Ww-A' wherein each -W-
is independently an Amino Acid unit; -Y- is a Spacer unit; w is an
integer ranging from 0 to 12; y is 0, 1 or 2; and -A' is selected
from
##STR00027## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IIi:
##STR00028## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.2 is
selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.3 is selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.11 is selected from
--H, --OH, --NH.sub.2, --NHR.sup.14, --N(R.sup.14).sub.2,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; R.sup.13 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); each
R.sup.14 is independently --H or --C.sub.1-C.sub.8 alkyl; R.sup.16
is -Yy-Ww-A' wherein each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit; w is an integer ranging from 0 to 12; y is 0,
1 or 2; and -A' is selected from
##STR00029## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
A compound of formula IIa-i or a pharmaceutically acceptable salt
or solvate thereof (a "Drug-Linker Compound") is useful for
treating cancer, an autoimmune disease or an infectious disease in
an animal or useful as an intermediate for the synthesis of a
Drug-Linker-Ligand Conjugate.
In another aspect, the present invention provides compositions
comprising an effective amount of a Drug-Linker-Ligand Conjugate
and a pharmaceutically acceptable carrier or vehicle.
In still another aspect, the present invention provides
compositions comprising an effective amount of a Drug-Linker
Compound and a pharmaceutically acceptable carrier or vehicle.
In yet another aspect, the present invention provides methods for
killing or inhibiting the multiplication of a tumor cell or cancer
cell, comprising administering to an animal in need thereof an
effective amount of a Drug-Linker Compound.
In another aspect, the present invention provides methods for
killing or inhibiting the multiplication of a tumor cell or cancer
cell, comprising administering to an animal in need thereof an
effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for
treating cancer, comprising administering to an animal in need
thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for treating
cancer, comprising administering to an animal in need thereof an
effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for killing
or inhibiting the replication of a cell that expresses an
auto-immune antibody, comprising administering to an animal in need
thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for killing or
inhibiting the replication of a cell that expresses an auto-immune
antibody, comprising administering to an animal in need thereof an
effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the invention provides methods for treating
an autoimmune disease, comprising administering to an animal in
need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for treating
an autoimmune disease, comprising administering to an animal in
need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
In still another aspect, the invention provides methods for
treating an infectious disease, comprising administering to an
animal in need thereof an effective amount of a Drug-Linker
Compound.
In still another aspect, the invention provides methods for
treating an infectious disease, comprising administering to an
animal in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
In yet another aspect, the present invention provides methods for
preventing the multiplication of a tumor cell or cancer cell,
comprising administering to an animal in need thereof an effective
amount of a Drug-Linker Compound.
In another aspect, the present invention provides methods for
preventing the multiplication of a tumor cell or cancer cell,
comprising administering to an animal in need thereof an effective
amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for
preventing cancer, comprising administering to an animal in need
thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for
preventing cancer, comprising administering to an animal in need
thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for
preventing the multiplication of a cell that expresses an
auto-immune antibody, comprising administering to an animal in need
thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for preventing
the multiplication of a cell that expresses an auto-immune
antibody, comprising administering to an animal in need thereof an
effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the invention provides methods for
preventing an autoimmune disease, comprising administering to an
animal in need thereof an effective amount of a Drug-Linker
Compound.
In yet another aspect, the invention provides methods for
preventing an autoimmune disease, comprising administering to an
animal in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
In still another aspect, the invention provides methods for
preventing an infectious disease, comprising administering to an
animal in need thereof an effective amount of a Drug-Linker
Compound.
In still another aspect, the invention provides methods for
preventing an infectious disease, comprising administering to an
animal in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
In another aspect, the invention provides a Drug-Linker Compound
which can be used as an intermediate for the synthesis of a
Drug-Linker-Ligand Conjugate.
The present invention may be understood more fully by reference to
the following detailed description, Figures and illustrative
examples, which are intended to exemplify non-limiting embodiments
of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the cytotoxicity of Compound 49 and Compound 53
against the H3396 cell line. Line -.DELTA.- represents Compound 49
and line -.smallcircle.- represents Compound 53.
FIG. 2 shows the cytotoxicity of Compounds 64, 65, 68 and 69
against the H3396 cell line. Line -.diamond-solid.- represents
Compound 64, line -.box-solid.- represents Compound 65, line
-.tangle-solidup.- represents Compound 68, and line -.times.-
represents Compound 69.
FIG. 3 shows the cytotoxicity of Compounds 64, 65, 68 and 69
against the HCT-116 cell line. Line -.diamond-solid.- represents
Compound 64, line -.box-solid.- represents Compound 65, line
-.tangle-solidup.- represents Compound 68, and line -.times.-
represents Compound 69.
FIG. 4 shows the cytotoxicity of Compounds 66 and 68 against the
H3396 cell line. Line -.quadrature.- represents Compound 66 and
line -*- represents Compound 68.
FIG. 5 shows the cytotoxicity of Compounds 66, 68 and 69 against
the Karpas human colorectal cell line. Line -.diamond-solid.-
represents Compound 66, line -.tangle-solidup.- represents Compound
68, and line -.times.- represents Compound 69.
FIG. 6 shows the cytotoxicity of Compounds 66 and 67 against the
H3396 cell line as a function of exposure length. The cells were
either exposed to the conjugates for the entire duration of the
assay without washing (96 hours), or were exposed to the conjugates
for 2 hours, washed, and then incubated for an additional 94 hours.
At the end of the 96 hour period, the cells were pulsed with Alamar
Blue to determine cell viability. Line - - represents Compound 66
at 2 h exposure, line -.smallcircle.- represents Compound 67 at 2 h
exposure, line -.cndot.- represents Compound 66 at 96 h exposure,
and line -.diamond.- represents Compound 67 at 96 h exposure.
FIG. 7 shows the effect of Compounds 66-69 on the growth of L2987
human lung adenocarcinoma xenograft tumors which were implanted in
nude mice. Line -.times.- represents untreated tumor, line --
represents Compound 66, line -.diamond-solid.- represents Compound
68, line -.gradient.- Compound 67, and line -.diamond.- represents
Compound 69.
FIG. 8 shows the effects of Compounds 66-69 on the growth of Karpas
human anaplastic large cell lymphoma xenograft tumors which were
implanted in nude mice. Line -.times.- represents untreated tumor,
line -.DELTA.- represents Compound 67, line -.cndot.- represents
Compound 69, line -.DELTA.- represents Compound 66, and line
-.smallcircle.- represents Compound 68.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Examples of an "animal" include, but are not limited to, a human,
rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat,
bird and fowl.
"Aryl" refers to a carbocyclic aromatic group Examples of aryl
groups include, but are not limited to, phenyl, naphthyl and
anthracenyl. A carbocyclic aromatic group or a heterocyclic
aromatic group can be unsubstituted or substituted with one or more
groups including, but not limited to, --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, --halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; where each R' is independently
selected from --C.sub.1-C.sub.8 alkyl and aryl.
The term "C.sub.1-C.sub.8 alkyl," as used herein refers to a
straight chain or branched, saturated or unsaturated hydrocarbon
having from 1 to 8 carbon atoms. Representative "C.sub.1-C.sub.8
alkyl" groups include, but are not limited to, -methyl, -ethyl,
-n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl,
-n-nonyl and -n-decyl; while branched C.sub.1-C.sub.8 alkyls
include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,
-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C.sub.1-C.sub.8
alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl,
-2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,
-3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl,
1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, -1-butynyl,
-2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1 butynyl.methyl,
ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,
n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl,
3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,
2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl,
2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl,
2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl,
2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl,
isoheptyl, n-octyl, and isooctyl. A C.sub.1-C.sub.8 alkyl group can
be unsubstituted or substituted with one or more groups including,
but not limited to, --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R', --S(O).sub.2R', --S(O)R',
--OH, -halogen, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and
--CN; where each R' is independently selected from
--C.sub.1-C.sub.8 alkyl and aryl.
A "C.sub.3-C.sub.8 carbocycle" is a 3-, 4-, 5-, 6-, 7- or
8-membered saturated or unsaturated non-aromatic carbocyclic ring.
Representative C.sub.3-C.sub.8 carbocycles include, but are not
limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl,
-cyclopentadienyl, -cyclohexyl, -cyclohexenyl,
-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,
-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and
-cyclooctadienyl. A C.sub.3-C.sub.8 carbocycle group can be
unsubstituted or substituted with one or more groups including, but
not limited to, --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R', --S(O).sub.2R', --S(O)R',
--OH, -halogen, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and
--CN; where each R' is independently selected from
--C.sub.1-C.sub.8 alkyl and aryl.
A "C.sub.3-C.sub.8 carbocyclo" refers to a C.sub.3-C.sub.8
carbocycle group defined above wherein one of the carbocycle groups
hydrogen atoms is replaced with a bond.
A "C.sub.1-C.sub.10 alkylene" is a straight chain, saturated
hydrocarbon group of the formula --(CH.sub.2).sub.1-10--. Examples
of a C.sub.1-C.sub.10 alkylene include methylene, ethylene,
propylene, butylene, pentylene, hexylene, heptylene, ocytylene,
nonylene and decalene.
An "arylene" is an aryl group which has two covalent bonds and can
be in the ortho, meta, or para configurations as shown in the
following structures:
##STR00030## in which the phenyl group can be unsubstituted or
substituted with up to four groups including, but not limited to,
--C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2--NHC(O)R', --S(O).sub.2R', --S(O)R', --OH,
--halogen, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and --CN;
where each R' is independently selected from --C.sub.1-C.sub.8
alkyl and aryl.
A "C.sub.3-C.sub.8 heterocycle" refers to an aromatic or
non-aromatic C.sub.3-C.sub.8 carbocycle in which one to four of the
ring carbon atoms are independently replaced with a heteroatom from
the group consisting of O, S and N. Representative examples of a
C.sub.3-C.sub.8 heterocycle include, but are not limited to,
benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl,
isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,
imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl,
pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl,
isoxazolyl and tetrazolyl. A C.sub.3-C.sub.8 Heterocycle can be
unsubstituted or substituted with up to seven groups including, but
not limited to, --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R', --S(O).sub.2R', --S(O)R',
--OH, -halogen, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and
--CN; where each R' is independently selected from
--C.sub.1-C.sub.8 alkyl and aryl.
"C.sub.3-C.sub.8 heterocyclo" refers to a C.sub.3-C.sub.8
heterocycle group defined above wherein one of the heterocycle
groups hydrogen atoms is replaced with a bond. A C.sub.3-C.sub.8
heterocyclo can be unsubstituted or substituted with up to six
groups including, but not limited to, --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; where each R' is independently
selected from --C.sub.1-C.sub.8 alkyl and aryl.
A "Compound of the Invention" is a Drug-Linker Compound or a
Drug-Linker-Ligand Conjugate.
In one embodiment, the Compounds of the Invention are in isolated
or purified form. As used herein, "isolated" means separated from
other components of (a) a natural source, such as a plant or animal
cell or cell culture, or (b) a synthetic organic chemical reaction
mixture. As used herein, "purified" means that when isolated, the
isolate contains at least 95%, preferably at least 98%, of a
Compound of the Invention by weight of the isolate.
Examples of a "Hydroxyl protecting group" include, but are not
limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether,
tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether,
trimethylsilyl ether, triisopropyl silyl ether, t-butyldimethyl
silyl ether, triphenylmethyl silyl ether, acetate ester,
substituted acetate esters, pivaloate, benzoate, methanesulfonate
and p-toluenesulfonate.
"Leaving group" refers to a functional group that can be
substituted by another functional group. Such leaving groups are
well known in the art, and examples include, but are not limited
to, a halide (e.g., chloride, bromide, iodide), methanesulfonyl
(mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl
(triflate), and trifluoromethylsulfonate.
The term "antibody," as used herein, refers to a full-length
immunoglobulin molecule or an immunologically active portion of a
full-length immunoglobulin molecule, i.e., a molecule that contains
an antigen binding site that immunospecifically binds an antigen of
a target of interest or part thereof, such targets including but
not limited to, cancer cell or cells that produce auto-immune
antibodies associated with an autoimmune disease. The
immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE,
IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1
and IgA2) or subclass of immunoglobulin molecule. The
immunoglobulins can be derived from any species. Preferably,
however, the immunoglobulin is of human, murine, or rabbit origin.
Antibodies useful in the invention are preferably monoclonal, and
include, but are not limited to, polyclonal, monoclonal,
bispecific, human, humanized or chimeric antibodies, single chain
antibodies, Fv, Fab fragments, F(ab') fragments, F(ab').sub.2
fragments, fragments produced by a Fab expression library,
anti-idiotypic (anti-Id) antibodies, CDR's, and epitope-binding
fragments of any of the above which immunospecifically bind to
cancer cell antigens, viral antigens or microbial antigens.
The phrase "pharmaceutically acceptable salt," as used herein,
refers to pharmaceutically acceptable organic or inorganic salts of
a Compound of the Invention. The Compounds of the Invention contain
at least one amino group, and accordingly acid addition salts can
be formed with this amino group. Preferred salts include, but are
not limited, to sulfate, citrate, acetate, oxalate, chloride,
bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate,
isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,
benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A
pharmaceutically acceptable salt may involve the inclusion of
another molecule such as an acetate ion, a succinate ion or other
counterion. The counterion may be any organic or inorganic moiety
that stabilizes the charge on the parent compound. Furthermore, a
pharmaceutically acceptable salt may have more than one charged
atom in its structure. Instances where multiple charged atoms are
part of the pharmaceutically acceptable salt can have multiple
counterions. Hence, a pharmaceutically acceptable salt can have one
or more charged atoms and/or one or more counterion.
"Pharmaceutically acceptable solvate" refers to an association of
one or more solvent molecules and a Compound of the Invention.
Examples of solvents that form pharmaceutically acceptable solvates
include, but are not limited to, water, isopropanol, ethanol,
methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
In the context of cancer, the term "treating" includes any or all
of: preventing growth of tumor cells or cancer cells, preventing
replication of tumor cells or cancer cells, lessening of overall
tumor burden and ameliorating one or more symptoms associated with
the disease.
In the context of an autoimmune disease, the term "treating"
includes any or all of: preventing replication of cells associated
with an autoimmune disease state including, but not limited to,
cells capable of producing an autoimmune antibody, lessening the
autoimmune-antibody burden and ameliorating one or more symptoms of
an autoimmune disease.
In the context of an infectious disease, the term "treating"
includes any or all of: preventing the growth, multiplication or
replication of the pathogen that causes the infectious disease and
ameliorating one or more symptoms of an infectious disease.
The following abbreviations are used herein and have the indicated
definitions: AE is auristatin E, Boc is N-(t-butoxycarbonyl), cit
is citrulline, dap is dolaproine, DCC is
1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA is
diethylamine, DEAD is diethylazodicarboxylate, DEPC is
diethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate,
DIEA is N,N-diisopropylethylamine, dil is dolaisoleuine, DMAP is
4-dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or
1,2-dimethoxyethane), DMF is N,N-dimethylformamide, DMSO is
dimethylsulfoxide, doe is dolaphenine, dov is N,N-dimethylvaline,
DTNB is 5,5'-dithiobis(2-nitrobenzoic acid), DTPA is
diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ
is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is
electrospray mass spectrometry, EtOAc is ethyl acetate, Fmoc is
N-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU is
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is high
pressure liquid chromatography, ile is isoleucine, lys is lysine,
MeCN is acetonitrile, MeOH is methanol, Mtr is
4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is
(1S,2R)-(+)-norephedrine, PAB is p-aminobenzyl, PBS is
phosphate-buffered saline (pH 7.4), PEG is polyethylene glycol, Ph
is phenyl, Pnp is p-nitrophenyl, MC is 6-maleimidocaproyl, Ph is
phenyl, phe is L-phenylalanine, PyBrop is
bromo-tris-pyrrolidino-phosphonium hexafluorophosphate, SEC is
size-exclusion chromatography, Su is succinimide, TFA is
trifluoroacetic acid, TLC is thin layer chromatography, UV is
ultraviolet, val is valine.
Drug-Linker-Ligand Conjugates
As stated above, the invention provides compounds of the formula
Ia:
##STR00031## and pharmaceutically acceptable salts and solvates
thereof wherein, L- is a Ligand unit; -A- is a Stretcher unit; a is
0 or 1; each -W- is independently an Amino Acid unit; -Y- is a
Spacer unit; w is an integer ranging from 0 to 12; y is 0, 1 or 2;
p ranges from 1 to about 20; and -D is a Drug unit of the
formula
##STR00032## wherein, independently at each location: R.sup.2 is
selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.3 is selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from
##STR00033## Z is --O--, --S--, --NH-- or --N(R.sup.14)--; R.sup.11
is selected from --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; R.sup.13 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); and each
R.sup.14 is independently --H or --C.sub.1-C.sub.8 alkyl.
In one embodiment R.sup.10 is selected from
##STR00034##
In another embodiment, w is an integer ranging from 2 to 12.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
Illustrative classes of compounds of formula Ia have the
structures:
##STR00035## ##STR00036## and pharmaceutically acceptable salts and
solvates thereof, where L- is a Ligand unit, E is --CH.sub.2-- or
--CH.sub.2CH.sub.2O--; e is an integer ranging either from 0-10
when E is --CH.sub.2--, or from 1-10 when E is
--CH.sub.2CH.sub.2--O--; F is --CH.sub.2--; f is 0 or 1; and p
ranges from 1 to about 20.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In another embodiment L is cBR96, cAC10 or 1F6.
Illustrative compounds of formula Ia have the structure:
##STR00037## ##STR00038## and pharmaceutically acceptable salts and
solvates thereof, where p ranges from about 7 to about 9.
In one embodiment p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In another embodiment, p is about 8.
In yet another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another aspect, the present invention provides compounds of
general formula Ib:
##STR00039## and pharmaceutically acceptable salts and solvates
thereof wherein, L- is a Ligand unit; -A- is a Stretcher unit; a is
0 or 1; each -W- is independently an Amino Acid unit; -Y- is a
Spacer unit; w is an integer ranging from 0 to 12; y is 0, 1 or 2;
p ranges from 1 to about 20; and -D is a Drug unit of the
formula
##STR00040## wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from
##STR00041## X is --O--, --S--, --NH-- or --N(R.sup.14)--, where X
is bonded to Y when y is 1 or 2, or X is bonded to W when y is 0; Z
is --O--, --S--, --NH-- or --N(R.sup.14)--; R.sup.11 is selected
from --H, --OH, --NH.sub.2, --NHR.sup.14, --N(R.sup.14).sub.2,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; R.sup.13 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); each
R.sup.14 is independently --H or --C.sub.1-C.sub.8 alkyl; and
R.sup.15 is -arylene-, --C.sub.3-C.sub.8 carbocyclo- or
--C.sub.3-C.sub.8 heterocyclo-.
In one embodiment, when R.sup.1 is --H, R.sup.10 is selected
from:
##STR00042##
In another embodiment, w is an integer ranging from 2 to 12.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
Illustrative classes of compounds of formula Ib have the
structure:
##STR00043## ##STR00044## pharmaceutically acceptable salts and
solvates thereof, where L- is Ligand unit, E is --CH.sub.2-- or
--CH.sub.2CH.sub.2O--; e is an integer ranging either from 0-10
when E is --CH.sub.2--, or 1-10 when E is --CH.sub.2CH.sub.2--O--;
F is --CH.sub.2--; f is 0 or 1; and p ranges from 1 to about
20.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another embodiment L is cBR96, cAC10 or 1F6.
Illustrative compounds of formula Ib have the structure:
##STR00045## and pharmaceutically acceptable salts and solvates
thereof, where p ranges from about 7 to about 9.
In one embodiment p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In another embodiment, p is about 8.
In yet another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another aspect, the present invention provides compounds of
general formula Ic:
##STR00046## L- is a Ligand unit; -A- is a Stretcher unit; a is 0
or 1; each -W- is independently an Amino Acid unit; w is an integer
ranging from 0 to 12; each n is independently 0 or 1; p ranges from
1 to about 20; and each -D is independently: (a) a Drug unit of the
formula:
##STR00047## wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from
##STR00048## X is --O--, --S--, --NH-- or --N(R.sup.14)--, where X
is bonded to --C(O)-- when y is 1 or 2, or X is bonded to
--CH.sub.2-- when n is 0; Z is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; R.sup.13 is selected from --H, --OH,
--NH.sub.2, --NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8
alkyl, --C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl),
-aryl, --C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); each
R.sup.14 is independently --H or --C.sub.1-C.sub.8 alkyl; and
R.sup.15 is -arylene-, --C.sub.3-C.sub.8 carbocyclo- or
--C.sub.3-C.sub.8 heterocyclo-; or (b) a Drug unit of the
formula:
##STR00049## wherein, independently at each location: R.sup.2 is
selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.3 is selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from
##STR00050## Z is --O--, --S--, --NH-- or --N(R.sup.14)--; R.sup.11
is selected from --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; R.sup.13 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); and each
R.sup.14 is independently --H or --C.sub.1-C.sub.8 alkyl.
In one embodiment, when the drug unit has the formula:
##STR00051## and R.sup.1 is --H, R.sup.10 is selected from
##STR00052##
In another embodiment, when the drug unit has the formula:
##STR00053## R.sup.10 is selected from
##STR00054##
In another embodiment, w is an integer ranging from 2 to 12.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
An illustrative compound of formula Ic has the structure:
##STR00055## wherein where L- is Ligand unit, E is --CH.sub.2-- or
--CH.sub.2CH.sub.2O--; e is an integer ranging either from 0-10
when E is --CH.sub.2--, or 1-10 when E is --CH.sub.2CH.sub.2--O--;
F is --CH.sub.2--; f is 0 or 1; and p ranges from 1 to about
20.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another embodiment L is cBR96, cAC10 or 1F6.
The Drug-Linker-Ligand Conjugates are useful for treating or
preventing cancer, an autoimmune disease or an infectious disease
in an animal.
It is understood that p is the average number of
-A.sub.a-W.sub.w-Y.sub.y-D units per ligand in a Drug-Linker-Ligand
Conjugate of formulas Ia, Ib and Ic.
In one embodiment p ranges from 1 to 15.
In another embodiment p ranges from 1 to 10.
In another embodiment, p ranges from 1 to about 8.
In a further embodiment p ranges from 1 to about 5.
In another embodiment p ranges from 1 to about 3.
In one embodiment p ranges from about 3 to about 5.
In one embodiment p ranges from about 7 to about 9.
In another embodiment p is about 8.
In yet another embodiment p is about 4.
In still another embodiment p is about 2.
The Drug-Linker-Ligand Conjugates of formulas Ia, Ib and Ic may
exist as mixtures, wherein each component of a mixture has a
different p value. For example, a Drug-Linker-Ligand Conjugate may
exist as a mixture of two separate Conjugates, one Conjugate
component wherein p is 7 and the other Conjugate component wherein
p is 8.
In one embodiment, a Drug-Linker-Ligand Conjugate exists as a
mixture of three separate conjugates wherein p for the three
separate conjugates is 1, 2, and 3, respectively.
In another embodiment, a Drug-Linker-Ligand Conjugate exists as a
mixture of three separate conjugates wherein p for the three
separate conjugates is 3, 4, and 5, respectively.
In another embodiment, a Drug-Linker-Ligand Conjugate exists as a
mixture of three separate conjugates wherein p for the three
separate conjugates is 5, 6, and 7, respectively.
In still another embodiment, a Drug-Linker-Ligand Conjugate exists
as a mixture of three separate conjugates wherein p for the three
separate conjugates is 7, 8, and 9, respectively.
In yet another embodiment, a Drug-Linker-Ligand Conjugate exists as
a mixture of three separate conjugates wherein p for the three
separate conjugates is 9, 10, and 11, respectively.
In still another embodiment, a Drug-Linker-Ligand Conjugate exists
as a mixture of three separate conjugates wherein p for the three
separate conjugates is 11, 12, and 13, respectively.
In another embodiment, a Drug-Linker-Ligand Conjugate exists as a
mixture of three separate conjugates wherein p for the three
separate conjugates is 13, 14, and 15, respectively.
Drug-Linker Compounds
The present invention provides compounds of the formula IIa:
##STR00056## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--, where X is bonded to Y when y is 1 or 2, or X is
bonded to W when y is 0; R.sup.11 is selected from --H, --OH,
--NH.sub.2, --NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8
alkyl, --C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl),
-aryl, --C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; each R.sup.14 is independently --H
or --C.sub.1-C.sub.8 alkyl; R.sup.16 is -Yy-Ww-A' wherein each -W-
is independently an Amino Acid unit; -Y- is a Spacer unit; w is an
integer ranging from 0 to 12; y is 0, 1 or 2; and -A' is selected
from
##STR00057## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
An illustrative compound of formula IIa has the structure:
##STR00058## and pharmaceutically acceptable salts and solvates
thereof.
In another aspect, the present invention provides compounds of the
formula IIb:
##STR00059## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; R.sup.13 is selected from hydrogen, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
alkyl-aryl, alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.14
is independently --H or --C.sub.1-C.sub.8 alkyl; R.sup.15 is
-arylene-, --C.sub.3-C.sub.8 carbocyclo- or --C.sub.3-C.sub.8
heterocyclo-; R.sup.16 is -Yy-Ww-A' wherein each -W- is
independently an Amino Acid unit; -Y- is a Spacer unit; w is an
integer ranging from 0 to 12; y is 0, 1 or 2; and -A' is selected
from
##STR00060## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IIc:
##STR00061## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; each R.sup.14 is independently --H
or --C.sub.1-C.sub.8 alkyl; R.sup.16 is -Yy-Ww-A' wherein each -W-
is independently an Amino Acid unit; -Y- is a Spacer unit; w is an
integer ranging from 0 to 12; y is 0, 1 or 2; and -A' is selected
from
##STR00062## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IId:
##STR00063## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; each R.sup.14 is independently --H
or --C.sub.1-C.sub.8 alkyl; R.sup.15 is -arylene-,
--C.sub.3-C.sub.8 carbocyclo- or --C.sub.3-C.sub.8 heterocyclo-;
R.sup.16 is -Yy-Ww-A' wherein each -W- is independently an Amino
Acid unit; -Y- is a Spacer unit; w is an integer ranging from 0 to
12; y is 0, 1 or 2; and -A' is selected from
##STR00064## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IIe:
##STR00065## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--; Z is --O--, --S--, --NH-- or --N(R.sup.14)--;
R.sup.11 is selected from --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; each R.sup.14 is independently --H or
--C.sub.1-C.sub.8 alkyl; R.sup.15 is -arylene-, --C.sub.3-C.sub.8
carbocyclo- or --C.sub.3-C.sub.8 heterocyclo-; R.sup.16 is
-Yy-Ww-A' wherein each -W- is independently an Amino Acid unit; -Y-
is a Spacer unit; w is an integer ranging from 0 to 12; y is 0, 1
or 2; and -A' is selected from
##STR00066## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IIf:
##STR00067## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; X is --O--, --S--, --NH-- or
--N(R.sup.14)--; Z is --O--, --S--, --NH-- or --N(R.sup.14)--;
R.sup.11 is selected from --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; each R.sup.14 is independently --H or
--C.sub.1-C.sub.8 alkyl; R.sup.15 is -arylene-, --C.sub.3-C.sub.8
carbocyclo- or --C.sub.3-C.sub.8 heterocyclo-; R.sup.16 is
-Yy-Ww-A' wherein each -W- is independently an Amino Acid unit; -Y-
is a Spacer unit; w is an integer ranging from 0 to 12; y is 0, 1
or 2; and -A' is selected from
##STR00068## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In one embodiment R.sup.1 is selected from --C.sub.1-C.sub.8 alkyl
and --C.sub.3-C.sub.8 carbocycle; and R.sup.2 is selected from --H
and --C.sub.1-C.sub.8 alkyl; or R.sup.1 and R.sup.2 join, have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached
Illustrative compounds of formula IIf have the structure:
##STR00069## and pharmaceutically acceptable salts and solvates
thereof
In another aspect, the present invention provides compounds of the
formula IIg:
##STR00070## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.2 is
selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.3 is selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; Z is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; each R.sup.14 is independently --H
or --C.sub.1-C.sub.8 alkyl; R.sup.16 is -Yy-Ww-A' wherein each -W-
is independently an Amino Acid unit; -Y- is a Spacer unit; w is an
integer ranging from 0 to 12; y is 0, 1 or 2; and -A' is selected
from
##STR00071## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IIh:
##STR00072## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.2 is
selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.3 is selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; Z is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; each R.sup.14 is independently --H
or --C.sub.1-C.sub.8 alkyl; R.sup.16 is -Yy-Ww-A' wherein each -W-
is independently an Amino Acid unit; -Y- is a Spacer unit; w is an
integer ranging from 0 to 12; y is 0, 1 or 2; and -A' is selected
from
##STR00073## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the
formula IIi:
##STR00074## and pharmaceutically acceptable salts and solvates
thereof wherein, independently at each location: R.sup.2 is
selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.3 is selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.11 is selected from
--H, --OH, --NH.sub.2, --NHR.sup.14, --N(R.sup.14).sub.2,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; R.sup.13 is selected from hydrogen, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
alkyl-aryl, alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.14
is independently --H or --C.sub.1-C.sub.8 alkyl; R.sup.16 is
-Yy-Ww-A' wherein each -W- is independently an Amino Acid unit; -Y-
is a Spacer unit; w is an integer ranging from 0 to 12; y is 0, 1
or 2; and -A' is selected from
##STR00075## wherein G is selected from --Cl, --Br, --I, --O-mesyl
and --O-tosyl; J is selected from --Cl, --Br, --I, --F, --OH,
--O--N-succinimide, --O-(4-nitrophenyl), --O-pentafluorophenyl,
--O-tetrafluorophenyl and --O--C(O)--OR.sup.18; R.sup.17 is
selected from --C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-, -arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; and R.sup.18 is --C.sub.1-C.sub.8 alkyl or -aryl.
Illustrative compounds of formula IIi have the structures:
##STR00076## ##STR00077## ##STR00078## and pharmaceutically
acceptable salts and solvates thereof
The compounds of formulas IIa-i are useful for treating or
preventing cancer, an autoimmune disease or an infectious disease
in an animal.
The Linker Unit
The Linker unit of the Drug-Linker-Ligand Conjugate links the Drug
unit and the Ligand unit and has the formula:
-A.sub.a-W.sub.w-Y.sub.y- wherein: -A- is a Stretcher unit; a is 0
or 1; each -W- is independently an Amino Acid unit; w is
independently an integer ranging from 0 to 12; -Y- is a Spacer
unit; and y is 0, 1 or 2. The Stretcher Unit
The Stretcher unit (-A-), when present, links a Ligand unit to an
amino acid unit (- W). In this regard a Ligand (L) has a functional
group that can form a bond with a functional group of a Stretcher.
Useful functional groups that can be present on a ligand, either
naturally or via chemical manipulation include, but are not limited
to, sulfhydryl (--SH), amino, hydroxyl, carboxy, the anomeric
hydroxyl group of a carbohydrate, and carboxyl. Preferred Ligand
functional groups are sulfhydryl and amino. Sulfhydryl groups can
be generated by reduction of an intramolecular disulfide bond of a
Ligand. Alternatively, sulfhydryl groups can be generated by
reaction of an amino group of a lysine moiety of a Ligand using
2-iminothiolane (Traut's reagent) or another sulfhydryl generating
reagent.
In one embodiment, the Stretcher unit forms a bond with a sulfur
atom of the Ligand unit. The sulfur atom can be derived from a
sulfhydryl group of a Ligand. Representative Stretcher units of
this embodiment are depicted within the square brackets of Formulas
(IIIa) and (IIIb), wherein L-, -W-, -Y-, -D, w and y are as defined
above and R.sup.17 is selected from --C.sub.1-C.sub.10 alkylene-,
--C.sub.3-C.sub.8 carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-,
-arylene-, --C.sub.1-C.sub.10 alkylene-arylene-,
-arylene-C.sub.1-C.sub.10 alkylene-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 carbocyclo)-, --(C.sub.3-C.sub.8
carbocyclo)-C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
heterocyclo-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
heterocyclo)-, --(C.sub.3-C.sub.8 heterocyclo)-C.sub.1-C.sub.10
alkylene-, --(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1-10.
##STR00079##
An illustrative Stretcher unit is that of formula (IIIa) where
R.sup.17 is --(CH.sub.2).sub.5--:
##STR00080##
Another illustrative Stretcher unit is that of formula (IIIa) where
R.sup.17 is --(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is
2:
##STR00081##
Still another illustrative Stretcher unit is that of formula (IIIb)
where R.sup.17 is --(CH.sub.2).sub.5--:
##STR00082##
In another embodiment, the Stretcher unit is linked to the Ligand
unit via a disulfide bond between a sulfur atom of the Ligand unit
and a sulfur atom of the Stretcher unit. A representative Stretcher
unit of this embodiment is depicted within the square brackets of
Formula (IV), wherein R.sup.17, L-, -W-, -Y-, -D, w and y are as
defined above.
##STR00083##
In yet another embodiment, the reactive group of the Stretcher
contains a reactive site that can form a bond with a primary or
secondary amino group of a Ligand. Example of these reactive sites
include, but are not limited to, activated esters such as
succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters,
tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl
chlorides, isocyanates and isothiocyanates. Representative
Stretcher units of this embodiment are depicted within the square
brackets of Formulas (Va) and (Vb), wherein --R.sup.17--, L-, -W-,
-Y-, -D, w and y are as defined above;
##STR00084##
In yet another aspect of the invention, the reactive group of the
Stretcher contains a reactive site that is reactive to a
carbohydrate's (--CHO) group that can be present on a Ligand. For
example, a carbohydrate can be mildly oxidized using a reagent such
as sodium periodate and the resulting (--CHO) unit of the oxidized
carbohydrate can be condensed with a Stretcher that contains a
functionality such as a hydrazide, an oxime, a primary or secondary
amine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate,
and an arylhydrazide such as those described by Kaneko, T. et al.
Bioconjugate Chem 1991, 2, 133-41. Representative Stretcher units
of this embodiment are depicted within the square brackets of
Formulas (VIa)-(VIc), wherein --R.sup.17--, L-, -W-, -Y-, -D, w and
y are as defined above.
##STR00085## The Amino Acid Unit
The Amino Acid unit (-W-), when present, links the Stretcher unit
to the Spacer unit if the Spacer unit is present, links the
Stretcher unit to the Drug unit if the Spacer unit is absent, and
links the Ligand unit to the Drug unit if the Stretcher unit and
Spacer unit are absent.
-W.sub.w- is a dipeptide, tripeptide, tetrapeptide, pentapeptide,
hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,
undecapeptide or dodecapeptide unit. Each -W- unit independently
has the formula denoted below in the square brackets, and w is an
integer ranging from 0 to 12:
##STR00086## wherein R.sup.19 is hydrogen, methyl, isopropyl,
isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, --CH.sub.2OH,
--CH(OH)CH.sub.3, --CH.sub.2CH.sub.2SCH.sub.3,
--CH.sub.2CONH.sub.2, --CH.sub.2COOH, --CH.sub.2CH.sub.2CONH.sub.2,
--CH.sub.2CH.sub.2COOH, --(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2,
--(CH.sub.2).sub.3NH.sub.2, --(CH.sub.2).sub.3NHCOCH.sub.3,
--(CH.sub.2).sub.3NHCHO, --(CH.sub.2).sub.4NHC(.dbd.NH)NH.sub.2,
--(CH.sub.2).sub.4NH.sub.2, --(CH.sub.2).sub.4NHCOCH.sub.3,
--(CH.sub.2).sub.4NHCHO, --(CH.sub.2).sub.3NHCONH.sub.2,
--(CH.sub.2).sub.4NHCONH.sub.2,
--CH.sub.2CH.sub.2CH(OH)CH.sub.2NH.sub.2, 2-pyridylmethyl-,
3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,
##STR00087##
The Amino Acid unit of the Compounds of the Invention can be
enzymatically cleaved by one or more enzymes, including a
tumor-associated protease, to liberate the Drug unit (-D), which in
one embodiment is protonated in vivo upon release to provide a Drug
(D).
Illustrative W.sub.w units are represented by formulas
(VII)-(IX):
##STR00088## wherein R.sup.20 and R.sup.21 are as follows:
TABLE-US-00001 R.sup.20 R.sup.21 benzyl (CH.sub.2).sub.4NH.sub.2;
methyl (CH.sub.2).sub.4NH.sub.2; isopropyl
(CH.sub.2).sub.4NH.sub.2; isopropyl (CH.sub.2).sub.3NHCONH.sub.2;
benzyl (CH.sub.2).sub.3NHCONH.sub.2; isobutyl
(CH.sub.2).sub.3NHCONH.sub.2; sec-butyl
(CH.sub.2).sub.3NHCONH.sub.2; ##STR00089##
(CH.sub.2).sub.3NHCONH.sub.2; benzyl methyl; and benzyl
(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2;
##STR00090## wherein R.sup.20, R.sup.21 and R.sup.22 are as
follows:
TABLE-US-00002 R.sup.20 R.sup.21 R.sup.22 benzyl benzyl
(CH.sub.2).sub.4NH.sub.2; isopropyl benzyl
(CH.sub.2).sub.4NH.sub.2; and H benzyl
(CH.sub.2).sub.4NH.sub.2;
##STR00091## wherein R.sup.20, R.sup.21, R.sup.22 and R.sup.23 are
as follows:
TABLE-US-00003 R.sup.20 R.sup.21 R.sup.22 R.sup.23 H benzyl
isobutyl H; and methyl isobutyl methyl isobutyl.
Preferred Amino Acid units include, but are not limited to, units
of formula (VII) where: R.sup.20 is benzyl and R.sup.21 is
--(CH.sub.2).sub.4NH.sub.2, R.sup.20 isopropyl and R.sup.21 is
--(CH.sub.2).sub.4NH.sub.2; R.sup.20 isopropyl and R.sup.21 is
--(CH.sub.2).sub.3NHCONH.sub.2. Another preferred Amino Acid unit
is a unit of formula (VIII) where R.sup.20 is benzyl, R.sup.21 is
benzyl, and R.sup.22 is --(CH.sub.2).sub.4NH.sub.2.
-W.sub.w- units useful in the present invention can be designed and
optimized in their selectivity for enzymatic cleavage by a
particular enzymes, for example, a tumor-associated protease. In
one embodiment, a -W.sub.w- unit is that whose cleavage is
catalyzed by cathepsin B, C and D, or a plasmin protease.
In one embodiment, -W.sub.w- is a dipeptide, tripeptide or
pentapeptide.
Where R.sup.19, R.sup.20, R.sup.21, R.sup.22 or R.sup.23 is other
than hydrogen, the carbon atom to which R.sup.19, R.sup.20,
R.sup.21, R.sup.22 or R.sub.23 is attached is chiral.
Each carbon atom to which R.sup.19, R.sup.20, R.sup.21, R.sup.22 or
R.sup.23 is attached is independently in the (S) or (R)
configuration.
The Spacer Unit
The Spacer unit (-Y-), when present, links an Amino Acid unit to
the Drug unit when an Amino Acid unit is present. Alternately, the
Spacer unit links the Stretcher unit to the Drug unit when the
Amino Acid unit is absent. The Spacer unit also links the Drug unit
to the ligand unit when both the Amino Acid unit and Stretcher unit
are absent. Spacer units are of two general types: self-immolative
and non self-immolative. A non self-immolative Spacer unit is one
in which part or all of the Spacer unit remains bound to the Drug
unit after cleavage, particularly enzymatic, of an Amino Acid unit
from the Drug-Linker-Ligand Conjugate or the Drug-Linker Compound.
Examples of a non self-immolative Spacer unit include, but are not
limited to a (glycine-glycine) Spacer unit and a glycine Spacer
unit (both depicted in Scheme 1). When a Compound of the Invention
containing a glycine-glycine Spacer unit or a glycine Spacer unit
undergoes enzymatic cleavage via a tumor-cell associated-protease,
a cancer-cell-associated protease or a lymphocyte-associated
protease, a glycine-glycine-Drug moiety or a glycine-Drug moiety is
cleaved from L-A.sub.a-W.sub.w-. In one embodiment, an independent
hydrolysis reaction takes place within the target cell, cleaving
the glycine-Drug unit bond and liberating the Drug.
In a preferred embodiment, -Y.sub.y- is a p-aminobenzyl alcohol
(PAB) unit (see Schemes 2 and 3) whose phenylene portion is
substituted with Q.sub.m where Q is is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; and m is
an integer ranging from 0-4.
##STR00092##
In one embodiment, a non self-immolative Spacer unit (-Y-) is
-Gly-Gly-.
In another embodiment, a non self-immolative the Spacer unit (-Y-)
is -Gly-.
In one embodiment, the invention provides a Drug-Linker Compound or
a Drug-Linker Ligand Conjugate in which the Spacer unit is absent
(y=0), or a pharmaceutically acceptable salt or solvate
thereof.
Alternatively, a Compound of the Invention containing a
self-immolative Spacer unit can release -D without the need for a
separate hydrolysis step. In this embodiment, -Y- is a PAB group
that is linked to -W.sub.w- via the amino nitrogen atom of the PAB
group, and connected directly to -D via a carbonate, carbamate or
ether group. Without being bound by theory, Scheme 2 depicts a
possible mechanism of Drug release of a PAB group which is attached
directly to -D via a carbamate or carbonate group.
##STR00093## where Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; m is an
integer ranging from 0-4; and p ranges from 1 to about 20.
Without being bound by theory, Scheme 3 depicts a possible
mechanism of Drug release of a PAB group which is attached directly
to -D via an ether or amine linkage.
##STR00094## where Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; m is an
integer ranging from 0-4; and p ranges from 1 to about 20.
Other examples of self-immolative spacers include, but are not
limited to, aromatic compounds that are electronically similar to
the PAB group such as 2-aminoimidazol-5-methanol derivatives (see
Hay et al., Bioorg. Med. Chem. Lett., 1999, 9, 2237) and ortho or
para-aminobenzylacetals. Spacers can be used that undergo
cyclization upon amide bond hydrolysis, such as substituted and
unsubstituted 4-aminobutyric acid amides (Rodrigues et al.,
Chemistry Biology, 1995, 2, 223), appropriately substituted
bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J.
Amer. Chem. Soc., 1972, 94, 5815) and 2-aminophenylpropionic acid
amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867).
Elimination of amine-containing drugs that are substituted at the
a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27,
1447) are also examples of self-immolative spacer useful in the
Compounds of the Invention.
In a preferred embodiment, the Spacer unit is a branched
bis(hydroxymethyl)styrene (BHMS) unit as depicted in Scheme 4,
which can be used to incorporate and release multiple drugs.
##STR00095## where Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; m is an
integer ranging from 0-4; n is 0 or 1; and p ranges raging from 1
to about 20.
In one embodiment, the -D moieties are the same.
In another embodiment, the -D moieties are different.
Preferred Spacer units (-Y.sub.y-) are represented by Formulas
(X)-(XII):
##STR00096## where Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; and m is
an integer ranging from 0-4;
##STR00097## The Drug Unit
-D is a Drug unit having a nitrogen or oxygen atom that can form a
bond with the Spacer unit when y=1 or 2 or with the C-terminal
carbonyl group of an Amino Acid unit when y=0.
In one embodiment, -D is represented by the formula:
##STR00098## wherein, independently at each location: R.sup.2 is
selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.3 is selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from
##STR00099## Z is --O--, --S--, --NH-- or --N(R.sup.14)--; R.sup.11
is selected from --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; R.sup.13 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); and each
R.sup.14 is independently --H or --C.sub.1-C.sub.8 alkyl.
In one embodiment, R.sup.10 is selected from
##STR00100##
In a preferred embodiment, -D has the formula
##STR00101## or a pharmaceutically acceptable salt or solvate
thereof, wherein, independently at each location: R.sup.2 is
selected from --H and -methyl; R.sup.3 is selected from --H,
-methyl, and -isopropyl; R.sup.4 is selected from --H and -methyl;
R.sup.5 is selected from -isopropyl, -isobutyl, -sec-butyl, -methyl
and -t-butyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- where R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl, and
--C.sub.3-C.sub.8 carbocycle, and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and -methyl; each R.sup.8 is
independently selected from --OH, -methoxy and -ethoxy; R.sup.10 is
selected from
##STR00102## R.sup.24 is selected from H and --C(O)R.sup.25;
wherein R.sup.25 is selected from --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.26 is selected from
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle, -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); Z is
--O--, --NH--, --OC(O)--, --NHC(O)--, --N(R.sup.28)C(O)--; where
R.sup.28 is selected from --H and --C.sub.1-C.sub.8 alkyl; n is 0
or 1; and R.sup.27 is selected from --H, --N.sub.3,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle, -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle) when n is
0; and R.sup.27 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) when n is 1.
In one embodiment, R.sup.10 is selected from
##STR00103##
In another embodiment, -D is represented by the formula:
##STR00104## wherein, independently at each location: R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; R.sup.3 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl; R.sup.7
is selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from --H, --OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle and --O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected
from --H and --C.sub.1-C.sub.8 alkyl; R.sup.10 is selected from
##STR00105## X is --O--, --S--, --NH-- or --N(R.sup.14)--, where X
forms a bond with a Linker unit; Z is --O--, --S--, --NH-- or
--N(R.sup.14)--; R.sup.11 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); or
R.sup.11 is an oxygen atom which forms a carbonyl unit (C.dbd.O)
with the carbon atom to which it is attached and a hydrogen atom on
this carbon atom is replaced by one of the bonds in the (C.dbd.O)
double bond; each R.sup.12 is independently selected from -aryl and
--C.sub.3-C.sub.8 heterocycle; R.sup.13 is selected from --H, --OH,
--NH.sub.2, --NHR.sup.14, --N(R.sup.14).sub.2,
--O--(C.sub.1-C.sub.8 alkyl), --C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.14 is independently
--H or --C.sub.1-C.sub.8 alkyl; and R.sup.15 is -arylene-,
--C.sub.3-C.sub.8 carbocyclo- or --C.sub.3-C.sub.8
heterocyclo-.
In one embodiment, when R.sup.1 is --H, R.sup.10 is selected
from:
##STR00106##
In a preferred embodiment, -D has the formula
##STR00107## or a pharmaceutically acceptable salt or solvate
thereof, wherein, independently at each location: R.sup.1 is
selected from --H and -methyl; R.sup.2 is selected from --H and
-methyl; R.sup.3 is selected from --H, -methyl, and -isopropyl;
R.sup.4 is selected from --H and -methyl; R.sup.5 is selected from
-isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl; or R.sup.4
and R.sup.5 join, have the formula --(CR.sup.aR.sup.b).sub.n--
where R.sup.a and R.sup.b are independently selected from --H,
--C.sub.1-C.sub.8 alkyl, and --C.sub.3-C.sub.8 carbocycle, and N is
selected from 2, 3, 4, 5 and 6, and form a ring with the carbon
atom to which they are attached; R.sup.6 is selected from --H and
-methyl; each R.sup.8 is independently selected from --OH, -methoxy
and -ethoxy; R.sup.10 is selected from
##STR00108##
where X is --O--, --NH-- or --N(R.sup.14)-- and forms a bond with Y
when y is 1 or 2, with W when y is 0, and with A when w and y are
both 0;
Z is --O--, --NH-- or --N(R.sup.14)--;
R.sup.13 is --H or -methyl;
R.sup.14 is C.sub.1-C.sub.8 alkyl; and
R.sup.15 is -arylene-, --C.sub.3-C.sub.8 carbocyclo or
--C.sub.3-C.sub.8 heterocyclo-,
In one embodiment, when R.sup.1 is -methyl, R.sup.10 is selected
from
##STR00109##
where X is --O--, --NH-- or --N(R.sup.14)-- and forms a bond with Y
when y is 1 or 2, and with W when y is 0;
Z is --O--, --NH-- or --N(R.sup.14)--;
R.sup.13 is --H or -methyl;
R.sup.14 is C.sub.1-C.sub.8 alkyl; and
R.sup.15 is -arylene-, --C.sub.3-C.sub.8 carbocyclo or
--C.sub.3-C.sub.8 heterocyclo-.
In another embodiment, when R.sup.1 is --H, R.sup.10 is selected
from:
##STR00110##
where X is --O--, --NH-- or --N(R.sup.14)-- and forms a bond with Y
when y is 1 or 2, and with W when y is 0;
Z is --O--, --NH-- or --N(R.sup.14)--;
R.sup.13 is --H or -methyl;
R.sup.14 is C.sub.1-C.sub.8 alkyl; and
R.sup.15 is -arylene-, --C.sub.3-C.sub.8 carbocyclo or
--C.sub.3-C.sub.8 heterocyclo-.
A Drug unit can form a bond with a Linker unit via a nitrogen atom
of a Drug's primary or secondary amino group, via an oxygen atom of
a Drug's hydroxyl group, or via a sulfur atom of a Drug's
sulfhydryl group to form a Drug-Linker Compound.
In a preferred embodiment, Drug units have the formula
##STR00111## ##STR00112## The Ligand Unit
The Ligand unit (L-) includes within its scope any unit of a Ligand
(L) that binds or reactively associates or complexes with a
receptor, antigen or other receptive moiety associated with a given
target-cell population. A Ligand can be any molecule that binds to,
complexes with or reacts with a moiety of a cell population sought
to be therapeutically or otherwise biologically modified. The
Ligand unit acts to deliver the Drug unit to the particular target
cell population with which the Ligand unit reacts. Such Ligands
include, but are not limited to, large molecular weight proteins
such as, for example, full-length antibodies, antibody fragments,
smaller molecular weight proteins, polypeptide or peptides, and
lectins.
A Ligand unit can form a bond to either a Stretcher unit or an
Amino Acid unit of a Linker. A Ligand unit can form a bond to a
Linker unit via a heteroatom of the Ligand. Heteroatoms that may be
present on a Ligand unit include sulfur (in one embodiment, from a
sulfhydryl group of a Ligand), oxygen (in one embodiment, from a
carbonyl, carboxyl or hydroxyl group of a Ligand) and nitrogen (in
one embodiment, from a primary or secondary amino group of a
Ligand). These heteroatoms can be present on the Ligand in the
Ligand's natural state, for example a naturally occurring antibody,
or can be introduced into the Ligand via chemical modification.
In a preferred embodiment, a Ligand has a sulfhydryl group and the
Ligand bonds to the Linker unit via the sulfhydryl group's sulfur
atom.
In another embodiment, the Ligand can have one or more carbohydrate
groups that can be chemically modified to have one or more
sulfhydryl groups. The Ligand unit bonds to the Stretcher unit via
the sulfhydryl group's sulfur atom.
In yet another embodiment, the Ligand can have one or more
carbohydrate groups that can be oxidized to provide an aldehyde
(--CHO) group (see Laguzza, et al., J. Med. Chem. 1989, 32(3),
548-55). The corresponding aldehyde can form a bond with a Reactive
Site on a Stretcher. Reactive sites on a Stretcher that can react
with a carbonyl group on a Ligand include, but are not limited to,
hydrazine and hydroxylamine.
Useful non-immunoreactive protein, polypeptide, or peptide Ligands
include, but are not limited to, transferrin, epidermal growth
factors ("EGF"), bombesin, gastrin, gastrin-releasing peptide,
platelet-derived growth factor, IL-2, IL-6, transforming growth
factors ("TGF"), such as TGF-.alpha. and TGF-.beta., vaccinia
growth factor ("VGF"), insulin and insulin-like growth factors I
and II, lectins and apoprotein from low density lipoprotein.
Useful Polyclonal antibody Ligands are heterogeneous populations of
antibody molecules derived from the sera of immunized animals.
Various procedures well known in the art may be used for the
production of polyclonal antibodies to an antigen-of-interest. For
example, for the production of polyclonal antibodies, various host
animals can be immunized by injection with an antigen of interest
or derivative thereof, including but not limited to rabbits, mice,
rats, and guinea pigs. Various adjuvants may be used to increase
the immunological response, depending on the host species, and
including but not limited to Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and corynebacterium parvum. Such adjuvants are
also well known in the art.
Useful monoclonal antibody Ligands are homogeneous populations of
antibodies to a particular antigen (e.g., a cancer cell antigen, a
viral antigen, a microbial antigen covalently linked to a second
molecule). A monoclonal antibody (mAb) to an antigen-of-interest
can be prepared by using any technique known in the art which
provides for the production of antibody molecules by continuous
cell lines in culture. These include, but are not limited to, the
hybridoma technique originally described by Kohler and Milstein
(1975, Nature 256, 495-497), the human B cell hybridoma technique
(Kozbor et al., 1983, Immunology Today 4: 72), and the
EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies
may be of any immunoglobulin class including IgG, IgM, IgE, IgA,
and IgD and any subclass thereof. The hybridoma producing the mAbs
of use in this invention may be cultivated in vitro or in vivo.
Useful monoclonal antibody Ligands include, but are not limited to,
human monoclonal antibodies or chimeric human-mouse (or other
species) monoclonal antibodies. Human monoclonal antibodies may be
made by any of numerous techniques known in the art (e.g., Teng et
al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80, 7308-7312; Kozbor et
al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982,
Meth. Enzymol. 92, 3-16).
The Ligand can also be a bispecific antibody. Methods for making
bispecific antibodies are known in the art. Traditional production
of full-length bispecific antibodies is based on the coexpression
of two immunoglobulin heavy chain-light chain pairs, where the two
chains have different specificities (Milstein et al., 1983, Nature
305:537-539). Because of the random assortment of immunoglobulin
heavy and light chains, these hybridomas (quadromas) produce a
potential mixture of 10 different antibody molecules, of which only
one has the correct bispecific structure. Purification of the
correct molecule, which is usually performed using affinity
chromatography steps, is rather cumbersome, and the product yields
are low. Similar procedures are disclosed in International
Publication No. WO 93/08829, and in Traunecker et al., EMBO J.
10:3655-3659 (1991).
According to a different and more preferred approach, antibody
variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2, and CH3 regions. It is preferred to have
the first heavy-chain constant region (CH1) containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
In a preferred embodiment of this approach, the bispecific
antibodies have a hybrid immunoglobulin heavy chain with a first
binding specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in
the other arm. This asymmetric structure facilitates the separation
of the desired bispecific compound from unwanted immunoglobulin
chain combinations, as the presence of an immunoglobulin light
chain in only one half of the bispecific molecule provides for a
facile way of separation (International Publication No. WO
94/04690) which is incorporated herein by reference in its
entirety.
For further details for generating bispecific antibodies see, for
example, Suresh et al., Methods in Enzymology, 1986, 121:210. Using
such techniques, bispecific antibody Ligands can be prepared for
use in the treatment or prevention of disease as defined
herein.
Bifunctional antibodies are also described, in European Patent
Publication No. EPA 0 105 360. As disclosed in this reference,
hybrid or bifunctional antibodies can be derived either
biologically, i.e., by cell fusion techniques, or chemically,
especially with cross-linking agents or disulfide-bridge forming
reagents, and may comprise whole antibodies or fragments thereof.
Methods for obtaining such hybrid antibodies are disclosed for
example, in International Publication WO 83/03679, and European
Patent Publication No. EPA 0 217 577, both of which are
incorporated herein by reference.
The Ligand can be a functionally active fragment, derivative or
analog of an antibody that immunospecifically binds to cancer cell
antigens, viral antigens, or microbial antigens. In this regard,
"Functionally active" means that the fragment, derivative or analog
is able to elicit anti-anti-idiotype antibodies that recognize the
same antigen that the antibody from which the fragment, derivative
or analog is derived recognized. Specifically, in a preferred
embodiment the antigenicity of the idiotype of the immunoglobulin
molecule can be enhanced by deletion of framework and CDR sequences
that are C-terminal to the CDR sequence that specifically
recognizes the antigen. To determine which CDR sequences bind the
antigen, synthetic peptides containing the CDR sequences can be
used in binding assays with the antigen by any binding assay method
known in the art (e.g., the BIA core assay)
Other useful Ligands include fragments of antibodies such as, but
not limited to, F(ab')2 fragments, which contain the variable
region, the light chain constant region and the CH1 domain of the
heavy chain can be produced by pepsin digestion of the antibody
molecule, and Fab fragments, which can be generated by reducing the
disulfide bridges of the F(ab')2 fragments. Other useful Ligands
are heavy chain and light chain dimers of antibodies, or any
minimal fragment thereof such as Fvs or single chain antibodies
(SCAs) (e.g., as described in U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or any
other molecule with the same specificity as the antibody.
Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are useful Ligands. A chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine monoclonal and a human immunoglobulin constant region.
(See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et
al., U.S. Pat. No. 4,816,397, which are incorporated herein by
reference in their entirety.) Humanized antibodies are antibody
molecules from non-human species having one or more complementarity
determining regions (CDRs) from the non-human species and a
framework region from a human immunoglobulin molecule. (See, e.g.,
Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by
reference in its entirety.) Such chimeric and humanized monoclonal
antibodies can be produced by recombinant DNA techniques known in
the art, for example using methods described in International
Publication No. WO 87/02671; European Patent Publication No.
184,187; European Patent Publication No. 171,496; European Patent
Publication No. 173,494; International Publication No. WO 86/01533;
U.S. Pat. No. 4,816,567; European Patent Publication No. 125,023;
Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc.
Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol.
139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA
84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et
al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl.
Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207;
Oi et al., 1986, BioTechniques 4:214; U.S. Pat. No. 5,225,539;
Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al., 1988, J. Immunol.
141:4053-4060; each of which is incorporated herein by reference in
its entirety.
Completely human antibodies are particularly desirable for Ligands.
Such antibodies can be produced using transgenic mice that are
incapable of expressing endogenous immunoglobulin heavy and light
chains genes, but which can express human heavy and light chain
genes. The transgenic mice are immunized in the normal fashion with
a selected antigen, e.g., all or a portion of a polypeptide of the
invention. Monoclonal antibodies directed against the antigen can
be obtained using conventional hybridoma technology. The human
immunoglobulin transgenes harbored by the transgenic mice rearrange
during B cell differentiation, and subsequently undergo class
switching and somatic mutation. Thus, using such a technique, it is
possible to produce therapeutically useful IgG, IgA, IgM and IgE
antibodies. For an overview of this technology for producing human
antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol.
13:65-93). For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., U.S. Pat. No.
5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S.
Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806; each of which is
incorporated herein by reference in its entirety. Other human
antibodies can be obtained commercially from, for example, Abgenix,
Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).
Completely human antibodies that recognize a selected epitope can
be generated using a technique referred to as "guided selection."
In this approach a selected non-human monoclonal antibody, e.g., a
mouse antibody, is used to guide the selection of a completely
human antibody recognizing the same epitope. (Jespers et al. (1994)
Biotechnology 12:899-903).
In other embodiments, the Ligand is a fusion protein of an
antibody, or a functionally active fragment thereof, for example in
which the antibody is fused via a covalent bond (e.g., a peptide
bond), at either the N-terminus or the C-terminus to an amino acid
sequence of another protein (or portion thereof, preferably at
least 10, 20 or 50 amino acid portion of the protein) that is not
the antibody. Preferably, the antibody or fragment thereof is
covalently linked to the other protein at the N-terminus of the
constant domain.
The Ligand antibodies include analogs and derivatives that are
either modified, i.e, by the covalent attachment of any type of
molecule as long as such covalent attachment permits the antibody
to retain its antigen binding immunospecificity. For example, but
not by way of limitation, the derivatives and analogs of the
antibodies include those that have been further modified, e.g., by
glycosylation, acetylation, pegylation, phosphylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular Ligand unit or other protein, etc.
Any of numerous chemical modifications can be carried out by known
techniques, including, but not limited to specific chemical
cleavage, acetylation, formylation, metabolic synthesis of
tunicamycin, etc. Additionally, the analog or derivative can
contain one or more unnaturalamino acids.
The Ligand antibodies include antibodies having modifications
(e.g., substitutions, deletions or additions) in amino acid
residues that interact with Fc receptors. In particular, the Ligand
antibodies include antibodies having modifications in amino acid
residues identified as involved in the interaction between the Fc
domain and the FcRn receptor (see, e.g., International Publication
No. WO 97/34631, which is incorporated herein by reference in its
entirety). Antibodies immunospecific for a cancer cell antigen can
be obtained commercially, for example, from Genentech (San
Francisco, Calif.) or produced by any method known to one of skill
in the art such as, e.g., chemical synthesis or recombinant
expression techniques. The nucleotide sequence encoding antibodies
immunospecific for a cancer cell antigen can be obtained, e.g.,
from the GenBank database or a database like it, the literature
publications, or by routine cloning and sequencing.
In a specific embodiment, known antibodies for the treatment or
prevention of cancer are used in accordance with the compositions
and methods of the invention. Antibodies immunospecific for a
cancer cell antigen can be obtained commercially or produced by any
method known to one of skill in the art such as, e.g., chemical
synthesis or recombinant expression techniques. The nucleotide
sequence encoding antibodies immunospecific for a cancer cell
antigen can be obtained, e.g., from the GenBank database or a
database like it, the literature publications, or by routine
cloning and sequencing. Examples of antibodies available for the
treatment of cancer include, but are not limited to, HERCEPTIN
(Trastuzumab; Genentech, CA) which is a humanized anti-HER2
monoclonal antibody for the treatment of patients with metastatic
breast cancer (Stebbing, J., Copson, E., and O'Reilly, S.
"Herceptin (trastuzamab) in advanced breast cancer" Cancer Treat
Rev. 26, 287-90, 2000); RITUXAN (rituximab; Genentech) which is a
chimeric anti-CD20 monoclonal antibody for the treatment of
patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation,
MA) which is a murine antibody for the treatment of ovarian cancer;
Panorex (Glaxo Wellcome, NC) which is a murine IgG.sub.2a antibody
for the treatment of colorectal cancer; BEC2 (ImClone Systems Inc.,
NY) which is murine IgG antibody for the treatment of lung cancer;
IMC-C225 (Imclone Systems Inc., NY) which is a chimeric IgG
antibody for the treatment of head and neck cancer; Vitaxin
(MedImmune, Inc., MD) which is a humanized antibody for the
treatment of sarcoma; Campath I/H (Leukosite, MA) which is a
humanized IgG.sub.1 antibody for the treatment of chronic
lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc.,
CA) which is a humanized IgG antibody for the treatment of acute
myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ) which
is a humanized IgG antibody for the treatment of non-Hodgkin's
lymphoma; Smart ID10 (Protein Design Labs, Inc., CA) which is a
humanized antibody for the treatment of non-Hodgkin's lymphoma;
Oncolym (Techniclone, Inc., CA) which is a murine antibody for the
treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA)
which is a humanized anti-CD2 mAb for the treatment of Hodgkin's
Disease or non-Hodgkin's lymphoma; anti-VEGF (Genentech, Inc., CA)
which is humanized antibody for the treatment of lung and
colorectal cancers; CEAcide (Immunomedics, NJ) which is a humanized
anti-CEA antibody for the treatment of colorectal cancer; IMC-1C11
(ImClone Systems, NJ) which is an anti-KDR chimeric antibody for
the treatment of colorectal cancer, lung cancers, and melanoma; and
Cetuximab (ImClone, NJ) which is an anti-EGFR chimeric antibody for
the treatment of epidermal growth factor positive cancers.
Other antibodies useful in the treatment of cancer include, but are
not limited to, antibodies against the following antigens: CA125
(ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6
(carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha
fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline
phosphatase (carcinomas), prostate specific antigen (prostate),
prostatic acid phosphatase (prostate), epidermal growth factor
(carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3
(carcinomas), MAGE-4 (carcinomas), anti-transferrin receptor
(carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA
(colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate),
IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's
lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human
chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40
(lymphoma), mucin (carcinomas), P21 (carcinomas), MPG (melanoma),
and Neu oncogene product (carcinomas). Some specific useful
antibodies include, but are not limited to, BR96 mAb (Trail, P. A.,
Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J.,
Casazza, A. M., Firestone, R. A., Hellstrom, I., Hellstrom, K. E.,
"Cure of Xenografted Human Carcinomas by BR96-Doxorubicin
Immunoconjugates" Science 1993, 261, 212-215), BR64 (Trail, P A,
Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J., Zoeckler, M.
E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza, A. M.,
Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R.
S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, M. B.,
Hellstrom, K. E., and Hellstrom, I. "Effect of Linker Variation on
the Stability, Potency, and Efficacy of Carcinoma-reactive
BR64-Doxorubicin Immunoconjugates" Cancer Research 1997, 57,
100-105, mAbs against the CD40 antigen, such as S2C6 mAb
(Francisco, J. A., Donaldson, K. L., Chace, D., Siegall, C. B., and
Wahl, A. F. "Agonistic properties and in vivo antitumor activity of
the anti-CD-40 antibody, SGN-14" Cancer Res. 2000, 60, 3225-3231),
mAbs against the CD70 antigen, such as 1F6 mAb, and mAbs against
the CD30 antigen, such as AC10 (Bowen, M. A., Olsen, K. J., Cheng,
L., Avila, D., and Podack, E. R. "Functional effects of CD30 on a
large granular lymphoma cell line YT" J. Immunol., 151, 5896-5906,
1993). Many other internalizing antibodies that bind to tumor
associated antigens can be used in this invention, and have been
reviewed (Franke, A. E., Sievers, E. L., and Scheinberg, D. A.,
"Cell surface receptor-targeted therapy of acute myeloid leukemia:
a review" Cancer Biother Radiopharm. 2000, 15, 459-76; Murray, J.
L., "Monoclonal antibody treatment of solid tumors: a coming of
age" Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel, S.,
Recombinant Antibodies, John Wiley, and Sons, New York, 1998).
In another specific embodiment, known antibodies for the treatment
or prevention of an autoimmune disease are used in accordance with
the compositions and methods of the invention. Antibodies
immunospecific for an antigen of a cell that is responsible for
producing autoimmune antibodies can be obtained from any
organization (e.g., a university scientist or a company such as
Genentech) or produced by any method known to one of skill in the
art such as, e.g., chemical synthesis or recombinant expression
techniques. In another embodiment, useful Ligand antibodies that
are immunospecific for the treatment of autoimmune diseases
include, but are not limited to, Anti-Nuclear Antibody; Anti ds
DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; Anti
Phospholipid Antibody IgM, IgG; Anti SM Antibody; Anti
Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody;
Thyroglobulin Antibody; Anti SCL-70; Anti-Jo; Anti-U.sub.1RNP;
Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti
Histones; Anti RNP; C-ANCA; P-ANCA; Anti centromere;
Anti-Fibrillarin, and Anti GBM Antibody.
In certain preferred embodiments, antibodies useful in the present
methods, can bind to both a receptor or a receptor complex
expressed on an activated lymphocyte. The receptor or receptor
complex can comprise an immunoglobulin gene superfamily member, a
TNF receptor superfamily member, an integrin, a cytokine receptor,
a chemokine receptor, a major histocompatibility protein, a lectin,
or a complement control protein. Non-limiting examples of suitable
immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19,
CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting
examples of suitable TNF receptor superfamily members are CD27,
CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK,
TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3,
TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins
are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c,
CD49d, CD49e, CD49f, CD103, and CD104. Non-limiting examples of
suitable lectins are C-type, S-type, and I-type lectin.
In one embodiment, the Ligand is an antibody that binds to an
activated lymphocyte that is associated with an autoimmune
disease.
In another specific embodiment, useful Ligand antibodies that are
immunospecific for a viral or a microbial antigen are monoclonal
antibodies. Preferably, Ligand antibodies that are immunospecific
for a viral antigen or microbial antigen are humanized or human
monoclonal antibodies. As used herein, the term "viral antigen"
includes, but is not limited to, any viral peptide, polypeptide
protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza
virus neuraminidase, influenza virus hemagglutinin, HTLV tax,
herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and
hepatitis B surface antigen) that is capable of eliciting an immune
response. As used herein, the term "microbial antigen" includes,
but is not limited to, any microbial peptide, polypeptide, protein,
saccharide, polysaccharide, or lipid molecule (e.g., a bacterial,
fungi, pathogenic protozoa, or yeast polypeptide including, e.g.,
LPS and capsular polysaccharide 5/8) that is capable of eliciting
an immune response.
Antibodies immunospecific for a viral or microbial antigen can be
obtained commercially, for example, from Genentech (San Francisco,
Calif.) or produced by any method known to one of skill in the art
such as, e.g., chemical synthesis or recombinant expression
techniques. The nucleotide sequence encoding antibodies that are
immunospecific for a viral or microbial antigen can be obtained,
e.g., from the GenBank database or a database like it, the
literature publications, or by routine cloning and sequencing.
In a specific embodiment, useful Ligand antibodies are those that
are useful for the treatment or prevention of viral or microbial
infection in accordance with the methods of the invention. Examples
of antibodies available useful for the treatment of viral infection
or microbial infection include, but are not limited to, SYNAGIS
(MedImmune, Inc., MD) which is a humanized anti-respiratory
syncytial virus (RSV) monoclonal antibody useful for the treatment
of patients with RSV infection; PRO542 (Progenics) which is a CD4
fusion antibody useful for the treatment of HIV infection; OSTAVIR
(Protein Design Labs, Inc., CA) which is a human antibody useful
for the treatment of hepatitis B virus; PROTOVIR (Protein Design
Labs, Inc., CA) which is a humanized IgG.sub.1 antibody useful for
the treatment of cytomegalovirus (CMV); and anti-LPS
antibodies.
Other antibodies useful in the treatment of infectious diseases
include, but are not limited to, antibodies against the antigens
from pathogenic strains of bacteria (Streptococcus pyogenes,
Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria
meningitidis, Corynebacterium diphtheriae, Clostridium botulinum,
Clostridium perfringens, Clostridium tetani, Hemophilus influenzae,
Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella
rhinoscleromotis, Staphylococcus aureus, Vibrio colerae,
Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio)
fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda,
Yersinia enterocolitica, Yersinia pestis, Yersinia
pseudotuberculosis, Shigella dysenteriae, Shigella flexneri,
Shigella sonnei, Salmonella typhimurium, Treponema pallidum,
Treponema pertenue, Treponema carateneum, Borrelia vincentii,
Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium
tuberculosis, Pneumocystis carinii, Francisella tularensis,
Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma
spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia
spp.); pathogenic fungi (Coccidioides immitis, Aspergillus
fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus
neoformans, Histoplasma capsulatum); protozoa (Entomoeba
histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas
hominis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma
rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania
tropica, Leishmania braziliensis, Pneumocystis pneumonia,
Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or
Helminiths (Enterobius vermicularis, Trichuris trichiura, Ascaris
lumbricoides, Trichinella spiralis, Strongyloides stercoralis,
Schistosoma japonicum, Schistosoma mansoni, Schistosoma
haematobium, and hookworms).
Other antibodies useful in this invention for treatment of viral
disease include, but are not limited to, antibodies against
antigens of pathogenic viruses, including as examples and not by
limitation: Poxviridae, Herpesviridae, Herpes Simplex virus 1,
Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae,
Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza
viruses, parainfluenza viruses, mumps, measles, respiratory
syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae,
Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis
E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae,
Rotoviridae, and Human Immunodeficiency Virus.
The antibodies suitable for use in the invention can be produced by
any method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or by recombinant expression, and
are preferably produced by recombinant expression techniques.
Production of Recombinant Antibodies
Ligand antibodies of the invention can be produced using any method
known in the art to be useful for the synthesis of antibodies, in
particular, by chemical synthesis or by recombinant expression, and
are preferably produced by recombinant expression techniques.
Recombinant expression of the Ligand antibodies, or fragment,
derivative or analog thereof, requires construction of a nucleic
acid that encodes the antibody. If the nucleotide sequence of the
antibody is known, a nucleic acid encoding the antibody may be
assembled from chemically synthesized oligonucleotides (e.g., as
described in Kutmeier et al., 1994, BioTechniques 17:242), which
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligation of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
Alternatively, a nucleic acid molecule encoding an antibody can be
generated from a suitable source. If a clone containing the nucleic
acid encoding the particular antibody is not available, but the
sequence of the antibody is known, a nucleic acid encoding the
antibody can be obtained from a suitable source (e.g., an antibody
cDNA library, or cDNA library generated from any tissue or cells
expressing the immunoglobulin) by PCR amplification using synthetic
primers hybridizable to the 3' and 5' ends of the sequence or by
cloning using an oligonucleotide probe specific for the particular
gene sequence.
If an antibody that specifically recognizes a particular antigen is
not commercially available (or a source for a cDNA library for
cloning a nucleic acid encoding such an immunoglobulin), antibodies
specific for a particular antigen can be generated by any method
known in the art, for example, by immunizing an animal, such as a
rabbit, to generate polyclonal antibodies or, more preferably, by
generating monoclonal antibodies, e.g., as described by Kohler and
Milstein (1975, Nature 256:495-497) or, as described by Kozbor et
al. (1983, Immunology Today 4:72) or Cole et al. (1985 in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96). Alternatively, a clone encoding at least the Fab portion of
the antibody can be obtained by screening Fab expression libraries
(e.g., as described in Huse et al., 1989, Science 246:1275-1281)
for clones of Fab fragments that bind the specific antigen or by
screening antibody libraries (See, e.g., Clackson et al., 1991,
Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA
94:4937).
Once a nucleic acid sequence encoding at least the variable domain
of the antibody is obtained, it can be introduced into a vector
containing the nucleotide sequence encoding the constant regions of
the antibody (see, e.g., International Publication No. WO 86/05807;
International Publication No. WO 89/01036; and U.S. Pat. No.
5,122,464). Vectors containing the complete light or heavy chain
that allow for the expression of a complete antibody molecule are
available. Then, the nucleic acid encoding the antibody can be used
to introduce the nucleotide substitutions or deletion necessary to
substitute (or delete) the one or more variable region cysteine
residues participating in an intrachain disulfide bond with an
amino acid residue that does not contain a sulfhydyl group. Such
modifications can be carried out by any method known in the art for
the introduction of specific mutations or deletions in a nucleotide
sequence, for example, but not limited to, chemical mutagenesis and
in vitro site directed mutagenesis (Hutchinson et al., 1978, J.
Biol. Chem. 253:6551).
In addition, techniques developed for the production of "chimeric
antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci.
81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et
al., 1985, Nature 314:452-454) by splicing genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity can be used. A chimeric antibody is a molecule in which
different portions are derived from different animal species, such
as those having a variable region derived from a murine monoclonal
antibody and a human immunoglobulin constant region, e.g.,
humanized antibodies.
Alternatively, techniques described for the production of single
chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science
242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be
adapted to produce single chain antibodies. Single chain antibodies
are formed by linking the heavy and light chain fragments of the Fv
region via an amino acid bridge, resulting in a single chain
polypeptide. Techniques for the assembly of functional Fv fragments
in E. coli may also be used (Skerra et al., 1988, Science
242:1038-1041).
Antibody fragments that recognize specific epitopes can be
generated by known techniques. For example, such fragments include,
but are not limited to, the F(ab')2 fragments that can be produced
by pepsin digestion of the antibody molecule and the Fab fragments
that can be generated by reducing the disulfide bridges of the
F(ab')2 fragments.
Once a nucleic acid sequence encoding a Ligand antibody has been
obtained, the vector for the production of the antibody can be
produced by recombinant DNA technology using techniques well known
in the art. Methods that are well known to those skilled in the art
can be used to construct expression vectors containing the antibody
coding sequences and appropriate transcriptional and translational
control signals. These methods include, for example, in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. See, for example, the techniques described
in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual,
2.sup.nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.) and Ausubel et al. (eds., 1998, Current Protocols in
Molecular Biology, John Wiley & Sons, NY).
An expression vector comprising the nucleotide sequence of an
antibody or the nucleotide sequence of an antibody can be
transferred to a host cell by conventional techniques (e.g.,
electroporation, liposomal transfection, and calcium phosphate
precipitation), and the transfected cells are then cultured by
conventional techniques to produce the antibody. In specific
embodiments, the expression of the antibody is regulated by a
constitutive, an inducible or a tissue, specific promoter.
The host cells used to express the recombinant Ligand antibody can
be either bacterial cells such as Escherichia coli, or, preferably,
eukaryotic cells, especially for the expression of whole
recombinant immunoglobulin molecule. In particular, mammalian cells
such as Chinese hamster ovary cells (CHO), in conjunction with a
vector such as the major intermediate early gene promoter element
from human cytomegalovirus is an effective expression system for
immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al.,
1990, BioTechnology 8:2).
A variety of host-expression vector systems can be utilized to
express the immunoglobulin Ligands. Such host-expression systems
represent vehicles by which the coding sequences of the antibody
can be produced and subsequently purified, but also represent cells
that can, when transformed or transfected with the appropriate
nucleotide coding sequences, express a Ligand immunoglobulin
molecule in situ. These include, but are not limited to,
microorganisms such as bacteria (e.g., E. coli and B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA or
cosmid DNA expression vectors containing immunoglobulin coding
sequences; yeast (e.g., Saccharomyces Pichia) transformed with
recombinant yeast expression vectors containing immunoglobulin
coding sequences; insect cell systems infected with recombinant
virus expression vectors (e.g., baculovirus) containing the
immunoglobulin coding sequences; plant cell systems infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing immunoglobulin coding sequences; or mammalian cell
systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g., metallothionein promoter) or
from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors can be
advantageously selected depending upon the use intended for the
antibody being expressed. For example, when a large quantity of
such a protein is to be produced, vectors that direct the
expression of high levels of fusion protein products that are
readily purified might be desirable. Such vectors include, but are
not limited, to the E. coli expression vector pUR278 (Ruther et
al., 1983, EMBO J. 2:1791), in which the antibody coding sequence
may be ligated individually into the vector in frame with the lac Z
coding region so that a fusion protein is produced; pIN vectors
(Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van
Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the
like. pGEX vectors can also be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In
general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption and binding to a matrix
glutathione-agarose beads followed by elution in the presence of
free glutathione. The pGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target gene
product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis
virus (AcNPV) or the analogous virus from Drosophila Melanogaster
is used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. The antibody coding sequence can be
cloned individually into non-essential regions (for example the
polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems
can be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest can be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene can then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) results in a
recombinant virus that is viable and capable of expressing the
immunoglobulin molecule in infected hosts. (e.g., see Logan &
Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific
initiation signals can also be required for efficient translation
of inserted antibody coding sequences. These signals include the
ATG initiation codon and adjacent sequences. Furthermore, the
initiation codon must be in phase with the reading frame of the
desired coding sequence to ensure translation of the entire insert.
These exogenous translational control signals and initiation codons
can be of a variety of origins, both natural and synthetic. The
efficiency of expression can be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., 1987, Methods in Enzymol.
153:51-544).
In addition, a host cell strain can be chosen to modulate the
expression of the inserted sequences, or modifies and processes the
gene product in the specific fashion desired. Such modifications
(e.g., glycosylation) and processing (e.g., cleavage) of protein
products can be important for the function of the protein.
Different host cells have characteristic and specific mechanisms
for the post-translational processing and modification of proteins
and gene products. Appropriate cell lines or host systems can be
chosen to ensure the correct modification and processing of the
foreign protein expressed. To this end, eukaryotic host cells that
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
can be used. Such mammalian host cells include, but are not limited
to, CHO, VERY, BH, Hela, COS, MDCK, 293, 293T, 3T3, W138, BT483,
Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins,
stable expression is preferred. For example, cell lines that stably
express an antibody can be engineered. Rather than using expression
vectors that contain viral origins of replication, host cells can
be transformed with DNA controlled by appropriate expression
control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker. Following the introduction of the foreign DNA,
engineered cells can be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The selectable
marker in the recombinant plasmid confers resistance to the
selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci that in turn can be cloned
and expanded into cell lines. This method can advantageously be
used to engineer cell lines which express the antibody Such
engineered cell lines can be particularly useful in screening and
evaluation of tumor antigens that interact directly or indirectly
with the antibody Ligand.
A number of selection systems can be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase
(Lowy et al., 1980, Cell 22:817) genes can be employed in tk-,
hgprt- or aprt-cells, respectively. Also, antimetabolite resistance
can be used as the basis of selection for the following genes:
dhfr, which confers resistance to methotrexate (Wigler et al.,
1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad.
Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993, TIB TECH 11(5):155-215) and hygro, which confers resistance
to hygromycin (Santerre et al., 1984, Gene 30:147). Methods
commonly known in the art of recombinant DNA technology which can
be used are described in Ausubel et al. (eds., 1993, Current
Protocols in Molecular Biology, John Wiley & Sons, NY;
Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al.
(eds), 1994, Current Protocols in Human Genetics, John Wiley &
Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).
The expression levels of an antibody can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use
of vectors based on gene amplification for the expression of cloned
genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press,
New York, 1987)). When a marker in the vector system expressing an
antibody is amplifiable, an increase in the level of inhibitor
present in culture of host cell will increase the number of copies
of the marker gene. Since the amplified region is associated with
the nucleotide sequence of the antibody, production of the antibody
will also increase (Crouse et al., 1983, Mol. Cell. Biol.
3:257).
The host cell can be co-transfected with two expression vectors of
the invention, the first vector encoding a heavy chain derived
polypeptide and the second vector encoding a light chain derived
polypeptide. The two vectors can contain identical selectable
markers that enable equal expression of heavy and light chain
polypeptides. Alternatively, a single vector can be used to encode
both heavy and light chain polypeptides. In such situations, the
light chain should be placed before the heavy chain to avoid an
excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52;
Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding
sequences for the heavy and light chains can comprise cDNA or
genomic DNA.
Once the antibody has been recombinantly expressed, it can be
purified using any method known in the art for purification of an
antibody, for example, by chromatography (e.g., ion exchange,
affinity, particularly by affinity for the specific antigen after
Protein A, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the
purification of proteins.
In a preferred embodiment, the Ligand is an antibody.
In a more preferred embodiment, the Ligand is a monoclonal
antibody.
In any case, the hybrid antibodies have a dual specificity,
preferably with one or more binding sites specific for the hapten
of choice or one or more binding sites specific for a target
antigen, for example, an antigen associated with a tumor, an
autoimmune disease, an infectious organism, or other disease
state.
Synthesis of the Compounds of the Invention
As described in more detail below, the Compounds of the Invention
are conveniently prepared using a Linker having two or more
Reactive Sites for binding to the Drug and Ligand. In one aspect of
the invention, a Linker has a Reactive site which has an
electrophilic group that is reactive to a nucleophilic group
present on a Ligand. Useful nucleophilic groups on a Ligand include
but are not limited to, sulfhydryl, hydroxyl and amino groups. The
heteroatom of the nucleophilic group of a Ligand is reactive to an
electrophilic group on a Linker and forms a covalent bond to a
Linker unit. Useful electrophilic groups include, but are not
limited to, maleimide and haloacetamide groups. The electrophilic
group provides a convenient site for Ligand attachment.
In another embodiment, a Linker has a Reactive site which has a
nucleophilic group that is reactive to an electrophilic group
present on a Ligand. Useful electrophilic groups on a Ligand
include, but are not limited to, aldehyde and ketone carbonyl
groups. The heteroatom of a nucleophilic group of a Linker can
react with an electrophilic group on a Ligand and form a covalent
bond to a Ligand unit. Useful nucleophilic groups on a Linker
include, but are not limited to, hydrazide, oxime, amino,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide. The electrophilic group on a Ligand provides a
convenient site for attachment to a Linker.
Carboxylic acid functional groups and chloroformate functional
groups are also useful reactive sites for a Linker because they can
react with primary or secondary amino groups of a Drug to form an
amide linkage. Also useful as a reactive site is a carbonate
functional group on a Linker which can react with an amino group or
hydroxyl group of a Drug to form a carbamate linkage or carbonate
linkage, respectively. Similarly, a Drug's phenol moiety can react
with the Linker, existing as an alcohol, under Mitsunobu
conditions.
Typically, peptide-based Drugs can be prepared by forming a peptide
bond between two or more amino acids and/or peptide fragments. Such
peptide bonds can be prepared, for example, according to the liquid
phase synthesis method (see E. Schroder and K. Lubke, "The
Peptides", volume 1, pp 76-136, 1965, Academic Press) that is well
known in the field of peptide chemistry.
In one embodiment, a Drug is prepared by combining about a
stoichiometric equivalent of a dipeptide and a tripeptide,
preferably in a one-pot reaction under suitable condensation
conditions. This approach is illustrated in the following Schemes
5-7. Thus, the tripeptide 6 can be prepared as shown in Scheme 5,
and the dipeptide 9 can be prepared as shown in Scheme 6. The two
fragments 6 and 9 can be condensed to provide a Drug 10 as shown in
Scheme 7.
The synthesis of an illustrative Stretcher having an electrophilic
maleimide group is illustrated in Schemes 8-9. General synthetic
methods useful for the synthesis of a Linker are described in
Scheme 10. Scheme 11 shows the construction of a Linker unit having
a val-cit group, an electrophilic maleimide group and a PAB
self-immolative Spacer group. Scheme 12 depicts the synthesis of a
Linker having a phe-lys group, an electrophilic maleimide group,
with and without the PAB self-immolative Spacer group. Scheme 13
presents a general outline for the synthesis of a Drug-Linker
Compound, while Scheme 14 presents an alternate route for preparing
a Drug-Linker Compound. Scheme 15 depicts the synthesis of a
branched linker containing a BHMS group. Scheme 16 outlines the
attachment of a Ligand to a Drug-Linker Compound to form a
Drug-Linker-Ligand Conjugate, and Scheme 17 illustrates the
synthesis of Drug-Linker-Ligand Conjugates having 2 or 4 drugs per
Ligand.
##STR00113##
As illustrated in Scheme 5, a protected amino acid 1 (where PG
represents an amine protecting group, R.sup.4 is selected from
hydrogen, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, alkyl-aryl,
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle,
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from H and methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from hydrogen, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and 6,
and form a ring with the carbon atom to which they are attached) is
coupled to t-butyl ester 2 (where R.sup.6 is selected from --H and
--C.sub.1-C.sub.8 alkyl; and R.sup.7 is selected from hydrogen,
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, alkyl-aryl,
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
alkyl-(C.sub.3-C.sub.8 heterocycle)) under suitable coupling
conditions, e.g., in the presence of PyBrop and
diisopropylethylamine, or using DCC (see, for example, Miyazaki, K.
et. al. Chem. Pharm. Bull. 1995, 43(10), 1706-1718).
Suitable protecting groups PG, and suitable synthetic methods to
protect an amino group with a protecting group are well known in
the art. See, e.g., Greene, T. W. and Wuts, P. G. M., Protective
Groups in Organic Synthesis, 2nd Edition, 1991, John Wiley &
Sons. Preferred protected amino acids 1 are PG-Ile and,
particularly, PG-Val, while other suitable protected amino acids
include, without limitation: PG-cyclohexylglycine,
PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid,
PG-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, and
PG-tert-butylglycine. Z is a preferred protecting group. Fmoc is
another preferred protecting group. A preferred t-butyl ester 2 is
dolaisoleuine t-butyl ester.
The dipeptide 3 can be purified, e.g., using chromatography, and
subsequently deprotected, e.g., using H.sub.2 and 10% Pd--C in
ethanol when PG is benzyloxycarbonyl, or using diethylamine for
removal of an Fmoc protecting group. The resulting amine 4 readily
forms a peptide bond with an amino acid 5 (where R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; and R.sup.3 is selected from hydrogen, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl),
-aryl, alkyl-aryl, alkyl-(C.sub.3-C.sub.8 carbocycle),
C.sub.3-C.sub.8 heterocycle and alkyl-(C.sub.3-C.sub.8
heterocycle)). N,N-Dialkyl amino acids are preferred amino acids 5,
such as commercially available N,N-dimethyl valine. Other
N,N-dialkyl amino acids can be prepared by reductive bis-alkylation
using known procedures (see, e.g., Bowman, R. E, Stroud, H. H J.
Chem. Soc., 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine
are two preferred amino acids 5 useful for the synthesis of
N-monoalkyl derivatives. The amine 4 and the amino acid 5 react to
provide the tripeptide 6 using coupling reagent DEPC with
triethylamine as the base.
Illustrative DEPC coupling methodology and the PyBrop coupling
methodology shown in Scheme 5 are outlined below in General
Procedure A and General Procedure B, respectively. Illustrative
methodology for the deprotection of a Z-protected amine via
catalytic hydrogenation is outlined below in General Procedure
C.
General Procedure A: Peptide synthesis using DEPC. The N-protected
or N,N-disubstituted amino acid or peptide 4 (1.0 eq.) and an amine
5 (1.1 eq.) are diluted with an aprotic organic solvent, such as
dichloromethane (0.1 to 0.5 M). An organic base such as
triethylamine or diisopropylethylamine (1.5 eq.) is then added,
followed by DEPC (1.1 eq.). The resulting solution is stirred,
preferably under argon, for up to 12 hours while being monitored by
HPLC or TLC. The solvent is removed in vacuo at room temperature,
and the crude product is purified using, for example, HPLC or flash
column chromatography (silica gel column). Relevant fractions are
combined and concentrated in vacuo to afford tripeptide 6 which is
dried under vacuum overnight.
General procedure B: Peptide synthesis using PyBrop. The amino acid
2 (1.0 eq.), optionally having a carboxyl protecting group, is
diluted with an aprotic organic solvent such as dichloromethane or
DME to provide a solution of a concentration between 0.5 and 1.0
mM, then diisopropylethylamine (1.5 eq.) is added. Fmoc-, or
Z-protected amino acid 1 (1.1 eq.) is added as a solid in one
portion, then PyBrop (1.2 eq.) is added to the resulting mixture.
The reaction is monitored by TLC or HPLC, followed by a workup
procedure similar to that described in General Procedure A.
General procedure C: Z-removal via catalytic hydrogenation.
Z-protected amino acid or peptide 3 is diluted with ethanol to
provide a solution of a concentration between 0.5 and 1.0 mM in a
suitable vessel, such as a thick-walled round bottom flask. 10%
palladium on carbon is added (5-10% w/w) and the reaction mixture
is placed under a hydrogen atmosphere. Reaction progress is
monitored using HPLC and is generally complete within 1-2 h. The
reaction mixture is filtered through a pre-washed pad of celite and
the celite is again washed with a polar organic solvent, such as
methanol after filtration. The eluent solution is concentrated in
vacuo to afford a residue which is diluted with an organic solvent,
preferably toluene. The organic solvent is then removed in vacuo to
afford the deprotected amine 4.
Table 1 lists representative examples of tripeptide intermediates
(compounds 39-43) that were prepared according to Scheme 5.
TABLE-US-00004 TABLE 1 ##STR00114## Compound X.sup.1 X.sup.2 39
Fmoc-N-Me-L-val L-val 40 Fmoc-N-Me-L-val L-ile 41 Fmoc-N-Me-gly
L-ile 42 dov L-val 43 dov L-ile .sup.adov =
N,N-dimethyl-L-valine
The dipeptide 9 can be readily prepared by condensation of the
modified amino acid Boc-Dolaproine 7 (see, for example, Pettit, G.
R., et al. Synthesis, 1996, 719-725), with (1S,2R)-norephedrine, L-
or D-phenylalaninol, or with synthetic p-acetylphenethylamine 8
(U.S. Pat. No. 3,445,518 to Shavel et al.) using condensing agents
well known for peptide chemistry, such as, for example, DEPC in the
presence of triethylamine, as shown in Scheme 6. Compound 7 may
also be condensed with commercially available compounds in this
manner to form dipeptides of formula 9. Examples of commercially
available compounds useful for this purpose include, but are not
limited to, norephedrine, ephedrine, and stereoisomers thereof
(Sigma-Sigma-Aldrich), L- or D-phenylalaninol (Sigma-Aldrich),
2-phenylethylamine (Sigma-Aldrich), 2-(4-aminophenyl)ethylamine
(Sigma-Aldrich), 1,2-ethanediamine-1,2-diphenyl (Sigma-Aldrich), or
4-(2-aminoethyl)phenol (Sigma-Aldrich), or with synthetically
prepared p-acetylphenethylamine, aryl- and heterocyclo-amides of
L-phenylalanine, 1-azidomethyl-2-phenylethylamine (prepared from
phenylalaninol according to a general procedure described in J.
Chem. Research (S), 1992, 391), and
1-(4-hydroxyphenyl)-2-phenylethylamine (European Patent Publication
No. 0356035 A2) among others.
##STR00115## where R.sup.8 is independently selected from --H,
--OH, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle and
--O--(C.sub.1-C.sub.8 alkyl); R.sup.9 is selected from --H and
--C.sub.1-C.sub.8 alkyl; and R.sup.10 is selected from:
##STR00116## where Z is --O--, --S--, --NH-- or --N(R.sup.14)--;
R.sup.11 is selected from --H, --OH, --NH.sub.2, --NHR.sup.14,
--N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8
carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C.sub.1-C.sub.8
alkyl-aryl, --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); or R.sup.11 is an oxygen atom
which forms a carbonyl unit (C.dbd.O) with the carbon atom to which
it is attached and a hydrogen atom on this carbon atom is replaced
by one of the bonds in the (C.dbd.O) double bond; each R.sup.12 is
independently selected from -aryl and --C.sub.3-C.sub.8
heterocycle; R.sup.13 is selected from --H, --OH, --NH.sub.2,
--NHR.sup.14, --N(R.sup.14).sub.2, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C.sub.1-C.sub.8 alkyl-aryl, --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), --C.sub.3-C.sub.8 heterocycle
and --C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); and each
R.sup.14 is independently --H or --C.sub.1-C.sub.8 alkyl.
Table 2 lists representative examples of dipeptides (Compounds
44-48) that were prepared according to Scheme 6.
TABLE-US-00005 TABLE 2 ##STR00117## Compound Y 44 ##STR00118## 45
##STR00119## 46 ##STR00120## 47 ##STR00121## 48 ##STR00122##
Scheme 7 illustrates a procedure useful for coupling tripeptide 6
and dipeptide 9 to form Drug 10. The coupling of 6 and 9 can be
accomplished using a strong acid, e.g. TFA, to facilitate Boc and
t-butyl ester cleavage, from dipeptide 9 and tripeptide 6,
respectively, followed by condensation conditions, e.g., utilizing
DEPC, or similar coupling reagent, in the presence of excess base
(triethylamine or equivalent) to provide Drug 10.
##STR00123##
An illustrative procedure for the synthesis of Drug 10 as depicted
in Scheme 7 is outlined below in General Procedure D.
The R.sup.10 group of a Drug of general formula 10 can be further
modified, if desired, to include a functional group that allows the
drug to be attached to a Linker. Examples of useful modifications
to the R.sup.10 group of a Drug 10, include, but are not limited to
the chemical transformations described below.
When R.sup.10 is
##STR00124## the hydroxyl group of R.sup.10 can be reacted with
commercially available or synthetically derived carboxylic acids or
carboxylic acid derivatives, including but not limited to,
carboxylic esters, acid chlorides, anhydrides and carbonates to
provide the corresponding esters according to well known methods in
the art. Coupling reagents, including, but not limited to DCC/DMAP
and EDCI/HOBt, can be useful in such coupling reactions between
alcohols and carboxylic acids or carboxylic acid derivatives. In a
preferred embodiment carboxylic acids are substituted or
unsubstituted aryl-carboxylic acids, for example, 4-aminobenzoic
acid. Thus, condensation of a hydroxyl group of the R.sup.10 group
shown above with carboxylic acids provides drugs of the general
structure 10 where R.sup.10 is
##STR00125## and where R.sup.11, R.sup.12, R.sup.14 and R.sup.15
are as previously described herein and X is selected from --OH,
--NH.sub.2 and --NHR.sup.14.
When R.sup.10 is
##STR00126## the azido group of the drug can be reduced (for an
example see J. Chem. Research (S), 1992, 391) to provide the
corresponding amino derivative wherein R.sup.10 is
##STR00127## the amino group of which can be reacted with the
carboxyl group of a carboxylic acid under general peptide coupling
conditions to provide drugs of general structure 10, where R.sup.10
is
##STR00128## and where R.sup.11, R.sup.12, R.sup.14 and R.sup.15
are as previously described herein and X is selected from --OH,
--NH.sub.2 and --NHR.sup.14.
Carboxylic acids useful in the above regard include, but are not
limited to, 4-aminobenzoic acid, p-acetylbenzoic acid and
2-amino-4-thiazolecarboxylic acid (Tyger Scientific, Inc., Ewing,
N.J.).
An Fmoc-protected amino group may be present on an amine-containing
R.sup.10 group of Drug 10 (e.g., as depicted in Table 2). The Fmoc
group is removable from the protected amine using diethylamine (see
General Procedure E as an illustrative example described
below).
General procedure D: Drug synthesis. A mixture of dipeptide 9 (1.0
eq.) and tripeptide 6 (1 eq.) is diluted with an aprotic organic
solvent, such as dichloromethane, to form a 0.1M solution, then a
strong acid, such as trifluoroacetic acid (1/2 v/v) is added and
the resulting mixture is stirred under a nitrogen atmosphere for
two hours at 0.degree. C. The reaction can be monitored using TLC
or, preferably, HPLC. The solvent is removed in vacuo and the
resulting residue is azeotropically dried twice, preferably using
toluene. The resulting residue is dried under high vacuum for 12 h
and then diluted with and aprotic organic solvent, such as
dichloromethane. An organic base such as triethylamine or
diisopropylethylamine (1.5 eq.) is then added, followed by either
PyBrop (1.2 eq.) or DEPC (1.2 eq.) depending on the chemical
functionality on the residue. The reaction mixture is monitored by
either TLC or HPLC and upon completion, the reaction is subjected
to a workup procedure similar or identical to that described in
General Procedure A.
General procedure E: Fmoc-removal using diethylamine. An
Fmoc-protected Drug 10 is diluted with an aprotic organic solvent
such as dichloromethane and to the resulting solution is added
diethylamine (1/2 v/v). Reaction progress is monitored by TLC or
HPLC and is typically complete within 2 h. The reaction mixture is
concentrated in vacuo and the resulting residue is azeotropically
dried, preferably using toluene, then dried under high vacuum to
afford Drug 10 having a deprotected amino group.
Thus, the above methods are useful for making Drugs that can be
used in the present invention.
To prepare a Drug-Linker Compound of the present invention, the
Drug is reacted with a reactive site on the Linker. In general, the
Linker can have the structure:
##STR00129## when both a Spacer unit (-Y-) and a Stretcher unit
(-A-) are present. Alternately, the Linker can have the
structure:
##STR00130## when the Spacer unit (-Y-) is absent.
The Linker can also have the structure:
##STR00131## when both the Stretcher unit (-A-) and the Spacer unit
(-Y-) are absent.
In general, a suitable Linker has an Amino Acid unit linked to an
optional Stretcher Unit and an optional Spacer Unit. Reactive Site
1 is present at the terminus of the Spacer and Reactive site 2 is
present at the terminus of the Stretcher. If a Spacer unit is not
present, then Reactive site 1 is present at the C-terminus of the
Amino Acid unit.
In one embodiment of the invention, Reactive Site No. 1 is reactive
to a nitrogen atom of the Drug, and Reactive Site No. 2 is reactive
to a sulfhydryl group on the Ligand. Reactive Sites 1 and 2 can be
reactive to different functional groups.
In one aspect of the invention, Reactive Site No. 1 is
##STR00132##
In another aspect of the invention, Reactive Site No. 1 is
##STR00133## wherein R is --Br, --Cl, --O-Su or
--O-(4-nitrophenyl).
In one embodiment, Reactive Site No. 1 is
##STR00134## wherein R is --Br, --Cl, --O-Su or
--O-(4-nitrophenyl), when a Spacer unit (-Y-) is absent.
Linkers having
##STR00135## at Reactive Site No. 1 where R is --Br or --Cl can be
prepared from Linkers having
##STR00136## at Reactive Site No. 1 by reacting the --COOH group
with PX.sub.3 or PX.sub.5, where X is --Br or --Cl. Alternatively,
linkers having
##STR00137## at Reactive Site No. 1 can be prepared from Linkers
having
##STR00138## at Reactive Site No. 1 by reacting the --COOH group
with thionyl chloride. For a general discussion of the conversion
of carboxylic acids to acyl halides, see March, Advanced Organic
Chemistry--Reactions, Mechanisms and Structure, 4.sup.th Ed., 1992,
John Wiley and Sons, New York, p. 437-438.
In another aspect of the invention, Reactive Site No. 1 is
##STR00139##
In still another aspect of the invention, Reactive Site No. 1
is
##STR00140## wherein R is --Cl, --O--CH(Cl)CCl.sub.3 or
--O-(4-nitrophenyl).
Linkers having
##STR00141## at Reactive Site No. 1 can be prepared from Linkers
having
##STR00142## at Reactive Site No. 1 by reacting the --OH group with
phosgene or triphosgene to form the corresponding chloroformate.
Linkers having
##STR00143## at Reactive Site No. 1 where R is --O--CH(Cl)CCl.sub.3
or --O-(4-nitrophenyl) can be prepared from Linkers having
##STR00144## at Reactive Site No. 1 by reacting the --OC(O)Cl group
with HO--CH(Cl)CCl.sub.3 or HO-(4-nitrophenyl), respectively. For a
discussion of this chemistry, see March, Advanced Organic
Chemistry--Reactions, Mechanisms and Structure, 4.sup.th Ed., 1992,
John Wiley and Sons, New York, p. 392.
In a further aspect of the invention, Reactive Site No. 1 is
##STR00145## wherein X is --F, --Cl, --Br, --I, or a leaving group
such as --O-mesyl, --O-tosyl or --O-triflate.
Linkers having
##STR00146## at Reactive Site No. 1 where X is --O-mesyl, --O-tosyl
and O-triflate can be prepared from Linkers having
##STR00147## at Reactive Site No. 1 by reacting the --OH group with
various reagents, including HCl, SOCl.sub.2, PCl.sub.5, PCl.sub.3
and POCl.sub.3 (where X is Cl); HBr, PBr.sub.3, PBr.sub.5 and
SOBr.sub.2 (where X is Br); HI (where X is I); and
CH.sub.3CH.sub.2NSF.sub.3 (DAST), SF.sub.4, SeF.sub.4 and
p-toluenesulfonyl fluoride (where X is F). For a general discussion
on the conversion of alcohols to alkyl halides, see March, Advanced
Organic Chemistry--Reactions, Mechanisms and Structure, 4.sup.th
Ed., 1992, John Wiley and Sons, New York, p. 431-433.
Linkers having
##STR00148## at Reactive Site No. 1 where X is --O-mesyl, --O-tosyl
and --O-triflate, can be prepared from Linkers having
##STR00149## at Reactive Site No. 1 by reacting the --OH group with
various mesylating, tosylating and triflating reagents,
respectively. Such reagents and methods for their use will be well
known to one of ordinary skill in the art of organic synthesis. For
a general discussion of mesyl, tosyl and triflates as leaving
groups, see March, Advanced Organic Chemistry--Reactions,
Mechanisms and Structure, 4.sup.th Ed., 1992, John Wiley and Sons,
New York, p. 353-354.
In one embodiment, when a Spacer unit (-Y-) is present, Reactive
Site No. 1 is
##STR00150## wherein R is --Cl, --O--CH(Cl)CCl.sub.3 or
--O-(4-nitrophenyl) and X is --F, --Cl, --Br, --I, or a leaving
group such as --O-mesyl, --O-tosyl or --O-triflate.
In another aspect of the invention, Reactive Site No. 1 is
##STR00151##
In still another aspect of the invention, Reactive Site No. 1 is a
p-nitrophenyl carbonate having the formula
##STR00152##
In one aspect of the invention, Reactive Site No. 2 is a
thiol-accepting group. Suitable thiol-accepting groups include
haloacetamide groups having the formula
##STR00153## where X represents a leaving group, preferably
O-mesyl, O-tosyl, --Cl, --Br, or --I; or a maleimide group having
the formula
##STR00154##
Useful Linkers can be obtained via commercial sources, such as
Molecular Biosciences Inc. (Boulder, Colo.), or synthesized in
accordance with procedures described in U.S. Pat. No. 6,214,345 to
Firestone et al., summarized in Schemes 8-10 below.
##STR00155## where X is --CH.sub.2-- or --CH.sub.2OCH.sub.2--; and
n is an integer ranging either from 0-10 when X is --CH.sub.2--; or
1-10 when X is --CH.sub.2OCH.sub.2--.
The method shown in Scheme 9 combines maleimide with a glycol under
Mitsunobu conditions to make a polyethylene glycol maleimide
Stretcher (see for example, Walker, M. A. J. Org. Chem. 1995, 60,
5352-5), followed by installation of a p-nitrophenyl carbonate
Reactive Site group.
##STR00156## where E is --CH.sub.2-- or --CH.sub.2OCH.sub.2--; and
e is an integer ranging from 0-8;
Alternatively, PEG-maleimide and PEG-haloacetamide stretchers can
be prepared as described by Frisch, et al., Bioconjugate Chem.
1996, 7, 180-186.
Scheme 10 illustrates a general synthesis of an illustrative Linker
unit containing a maleimide Stretcher group and optionally a
p-aminobenzyl ether self-immolative Spacer.
##STR00157## where Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen,-nitro or -cyano; m is an
integer ranging from 0-4; and n is an integer ranging from
0-10.
Useful Stretchers may be incorporated into a Linker using the
commercially available intermediates from Molecular Biosciences
(Boulder, Colo.) described below by utilizing known techniques of
organic synthesis.
Stretchers of formula (IIIa) can be introduced into a Linker by
reacting the following intermediates with the N-terminus of an
Amino Acid unit as depicted in Schemes 11 and 12:
##STR00158## where n is an integer ranging from 1-10 and T is --H
or --SO.sub.3Na;
##STR00159## where n is an integer ranging from 0-3;
##STR00160##
Stretcher units of formula (IIIb) can be introduced into a Linker
by reacting the following intermediates with the N-terminus of an
Amino Acid unit:
##STR00161## where X is --Br or --I; and
##STR00162##
Stretcher units of formula (IV) can be introduced into a Linker by
reacting the following intermediates with the N-terminus of an
Amino Acid unit:
##STR00163##
Stretcher units of formula (Va) can be introduced into a Linker by
reacting the following intermediates with the N-terminus of an
Amino Acid unit:
##STR00164##
Other Stretchers useful in the invention may be synthesized
according to known procedures. Aminooxy Stretchers of the formula
shown below can be prepared by treating alkyl halides with
N-Boc-hydroxylamine according to procedures described in Jones, D.
S. et al., Tetrahedron Letters, 2000, 41(10), 1531-1533; and Gilon,
C. et al., Tetrahedron, 1967, 23(11), 4441-4447.
##STR00165## where --R.sup.17-- is selected from --C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 carbocyclo-, --O--(C.sub.1-C.sub.8
alkyl)-, -arylene-, --C.sub.1-C.sub.10 alkylene-arylene-,
-arylene-C.sub.1-C.sub.10 alkylene-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 carbocyclo)-, --(C.sub.3-C.sub.8
carbocyclo)-C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
heterocyclo-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
heterocyclo)-, --(C.sub.3-C.sub.8 heterocyclo)-C.sub.1-C.sub.10
alkylene-, --(CH.sub.2CH.sub.2O).sub.r--,
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1-10;
Isothiocyanate Stretchers of the formula shown below may be
prepared from isothiocyanatocarboxylic acid chlorides as described
in Angew. Chem., 1975, 87(14), 517.
##STR00166## where --R.sup.17-- is as described herein.
Scheme 11 shows a method for obtaining of a val-cit dipeptide
Linker having a maleimide Stretcher and optionally a p-aminobenzyl
self-immolative Spacer.
##STR00167## where Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; and m is
an integer ranging from 0-4.
Scheme 12 illustrates the synthesis of a phe-lys(Mtr) dipeptide
Linker unit having a maleimide Stretcher unit and a p-aminobenzyl
self-immolative Spacer unit. Starting material 23 (lys(Mtr)) is
commercially available (Bachem, Torrance, Calif.) or can be
prepared according to Dubowchik, et al. Tetrahedrom Letters 1997,
38, 5257-60.
##STR00168## where Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; and m is
an integer ranging from 0-4.
As shown in Scheme 13, a Linker can be reacted with an amino group
of a Drug 10 to form a Drug-Linker Compound that contains an amide
or carbamate group, linking the Drug unit to the Linker unit. When
Reactive Site No. 1 is a carboxylic acid group, as in Linker 29,
the coupling reaction can be performed using HATU or PyBrop and an
appropriate amine base, resulting in a Drug-Linker Compound 30,
containing a amide bond between the Drug unit and the Linker unit.
When Reactive Site No. 1 is a carbonate, as in Linker 31, the
Linker can be coupled to the Drug using HOBt in a mixture of
DMF/pyridine to provide a Drug-Linker Compound 32, containing a
carbamate bond between the Drug unit and the Linker unit.
When Reactive Site No. 1 is an hydroxyl group, such as Linker 33,
the Linker can be coupled with a phenol group of a Drug using
Mitsunobu chemistry to provide a Drug-Linker Compound 34 having an
ether linkage between the Drug unit and the Linker unit.
Alternately, when Reactive Site No. 1 is a good leaving group, such
as in Linker 70, the Linker can be coupled with a hydroxyl group or
an amine group of a Drug via a nucleophilic substitution process to
provide a Drug-Linker Compound having an ether linkage (34) or an
amine linkage (71) between the Drug unit and the Linker unit.
Illustrative methods useful for linking a Drug to a Ligand to form
a Drug-Linker Compound are depicted in Scheme 13 and are outlined
in General Procedures G-J.
##STR00169##
General Procedure G: Amide formation using HATU. A Drug 10 (1.0
eq.) and an N-protected Linker containing a carboxylic acid
Reactive site (1.0 eq.) are diluted with a suitable organic
solvent, such as dichloromethane, and the resulting solution is
treated with HATU (1.5 eq.) and an organic base, preferably
pyridine (1.5 eq.). The reaction mixture is allowed to stir under
an inert atmosphere, preferably argon, for 6 h, during which time
the reaction mixture is monitored using HPLC. The reaction mixture
is concentrated and the resulting residue is purified using HPLC to
yield the amide 30.
General Procedure H: Carbamate formation using HOBt. A mixture of a
Linker 31 having a p-nitrophenyl carbonate Reactive site (1.1 eq.)
and Drug 10 (1.0 eq.) are diluted with an aprotic organic solvent,
such as DMF, to provide a solution having a concentration of 50-100
mM, and the resulting solution is treated with HOBt (2.0 eq.) and
placed under an inert atmosphere, preferably argon. The reaction
mixture is allowed to stir for 15 min, then an organic base, such
as pyridine (1/4 v/v), is added and the reaction progress is
monitored using HPLC. The Linker is typically consumed within 16 h.
The reaction mixture is then concentrated in vacuo and the
resulting residue is purified using, for example, HPLC to yield the
carbamate 32.
General Procedure I: Ether formation using Mitsunobu chemistry. A
Drug of general formula 10, which contains a free hydroxyl group,
is diluted with THF to make a 1.0 M solution and to this solution
is added a Linker (1.0 eq) containing an hydroxy group at Reactive
site No. 1 (33), followed by triphenylphosphine (1.5 eq.). The
reaction mixture is put under an argon atmosphere and cooled to
0.degree. C. DEAD (1.5 eq.) is then added dropwise via syringe and
the reaction is allowed to stir at room temperature while being
monitored using HPLC. The reaction is typically complete in 0.5-12
h, depending on the substrates. The reaction mixture is diluted
with water (in volume equal to that of the THF) and the reaction
mixture is extracted into EtOAc. The EtOAc layer is washed
sequentially with water and brine, then dried over MgSO4 and
concentrated. The resulting residue is purified via flash column
chromatography using a suitable eluent to provide ether 34.
General Procedure J: Ether/amine Formation via Nucleophilic
Substitution. A Drug of general formula 10, which contains a free
hydroxyl group or a free amine group, is diluted with a polar
aprotic solvent, such as THF, DMF or DMSO, to make a 1.0 M solution
and to this solution is added a non-nucleophilic base (about 1.5
eq), such as pyridine, diisopropylethylamine or triethylamine. The
reaction mixture is allowed to stir for about 1 hour, and to the
resulting solution is added an approximately 1.0M solution of
Linker 70 in a polar aprotic solvent, such as THF, DMF or DMSO. The
resulting reaction is stirred under an inert atmosphere while being
monitored using TLC or HPLC. The reaction is typically complete in
0.5-12 h, depending on the substrates. The reaction mixture is
diluted with water (in volume equal to that of the reaction volume)
and extracted into EtOAc. The EtOAc layer is washed sequentially
with water, 1N HCl, water, and brine, then dried over MgSO4 and
concentrated. The resulting residue is purified via flash column
chromatography using a suitable eluent to provide an ether of
formula 34 or an amine of formula 71, depending on whether the drug
10 contained a free hydroxyl group or a free amine group.
An alternate method of preparing Drug-Linker Compounds of the
invention is outlined in Scheme 14. Using the method of Scheme 14,
the Drug is attached to a partial Linker unit (19a, for example),
which does not have a Stretcher unit attached. This provides
intermediate 35, which has an Amino Acid unit having an
Fmoc-protected N-terminus. The Fmoc group is then removed and the
resulting amine intermediate 36 is then attached to a Stretcher
unit via a coupling reaction catalyzed using PyBrop or DEPC. The
construction of Drug-Linker Compounds containing either a
bromoacetamide Stretcher 39 or a PEG maleimide Stretcher 38 is
illustrated in Scheme 14.
##STR00170## where Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; and m is
an integer ranging from 0-4.
Methodology useful for the preparation of a Linker unit containing
a branched spacer is shown in Scheme 15.
##STR00171##
Scheme 15 illustrates the synthesis of a val-cit dipeptide linker
having a maleimide Stretcher unit and a bis(4-hydroxymethyl)styrene
(BHMS) unit. The synthesis of the BHMS intermediate (75) has been
improved from previous literature procedures (see International
Publication No, WO 9813059 to Firestone et al., and Crozet, M. P.;
Archaimbault, G.; Vanelle, P.; Nouguier, R. Tetrahedron Lett. 1985,
26, 5133-5134) and utilizes as starting materials, commercially
available diethyl(4-nitrobenzyl)phosphonate (72) and commercially
available 2,2-dimethyl-1,3-dioxan-5-one (73). Linkers 77 and 79 can
be prepared from intermediate 75 using the methodology described in
Scheme 11.
Scheme 16 illustrates methodology useful for making
Drug-Linker-Ligand conjugates of the invention having about 2 to
about 4 drugs per antibody.
##STR00172##
General Procedure K: Preparation of Conjugates having about 2 to
about 4 drugs per antibody.
Partial Reduction of the Antibody
In general, to prepare conjugates having 2 drugs per antibody, the
relevant antibody is reduced using a reducing agent such as
dithiothreitol (DTT) or tricarbonyl ethylphosphine (TCEP) (about
1.8 equivalents) in PBS with 1 mM DTPA, adjusted to pH 8 with 50 mM
borate. The solution is incubated at 37.degree. C. for 1 hour,
purified using a 50 ml G25 desalting column equilibrated in PBS/1
mM DTPA at 4.degree. C. The thiol concentration can be determined
according to General Procedure M, the protein concentration can be
determined by dividing the A280 value by 1.58 extinction
coefficient (mg/ml), and the ratio of thiol to antibody can be
determined according to General Procedure N.
Conjugates having 4 drugs per antibody can be made using the same
methodology, using about 4.2 equivalents of a suitable reducing
agent to partially reduce the antibody.
Conjugation of Drug-Linker to Partially Reduced Antibody
The partially reduced antibody samples can be conjugated to a
corresponding Drug-Linker compound using about 2.4 and about 4.6
molar equivalents of Drug-Linker compound per antibody to prepare
the 2 and 4 drug per antibody conjugates, respectively. The
conjugation reactions are incubated on ice for 1 hour, quenched
with about 20-fold excess of cysteine to drug, and purified by
elution over a G25 desalting column at about 4.degree. C. The
resulting Drug-Linker-Ligand conjugates are concentrated to about 3
mg/ml, sterile filtered, aliquoted and stored frozen.
Scheme 17 depicts the construction of a Drug-Linker-Ligand
Conjugate by reacting the sulfhydryl group of a Ligand with a
thiol-acceptor group on the Linker group of a Drug-Linker
Compound.
##STR00173##
Illustrative methods for attaching a Ligand antibody to a
Drug-Linker Compound are outlined below in General Procedures
L-R.
General Procedure L: Attachment of an Antibody Ligand to a
Drug-Linker Compound. All reaction steps are typically carried out
at 4.degree. C. Where the Ligand is a monoclonal antibody having
one or more disulfide bonds, solutions of the monoclonal antibody
(5-20 mg/mL) in phosphate buffered saline, pH 7.2, are reduced with
dithiothreitol (10 mM final) at 37.degree. C. for 30 minutes (See
General Procedure M) and separation of low molecular weight agents
is achieved by size exclusion chromatography on Sephadex G25
columns in PBS containing 1 mM diethylenetriaminepentaacetic
acid.
The sulfhydryl content in the Ligand can be determined using
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as described in General
Procedure M (see Riddles, P. W., Blakeley, R. L., and Zerner, B.
(1979) Anal. Biochem. 94, 75-81). To a PBS solution of Ligand
reduced according to General Procedure L, a Drug-Linker Compound in
MeCN is added so that the solution is 20% MeCN/PBS (vol/vol). The
amount of Drug-Linker Compound is approximately 10% more than the
total number of sulfhydryl groups on a Ligand. After 60 min at
4.degree. C., cysteine is added (20-fold excess over concentration
of the Drug-Linker Compound), the solution is concentrated by
ultrafiltration, and any low molecular weight agents are removed by
gel filtration. The number of Drug-Linker Compounds per antibody is
determined by uv/vis spectroscopy using formulas derived from the
relative extinction coefficients of the Ligands and Drug-Linker
Compounds as described in General Procedure O. The amount of
quenched Drug-Linker Compound is then determined as described in
General Procedure P using reverse-phase HPLC. The aggregation state
of the Ligand Antibodies of the Drug-Linker-Ligand Conjugates can
be determined using size-exclusion HPLC as described in General
Procedure R. The Drug-Linker-Ligand Conjugates can be used without
further purification.
General Procedure M: Reduction of the interchain disulfide bonds of
an Antibody. To a solution of 24 mg of an antibody (2.4 mL of 10
mg/mL solution) in suitable buffer is added 300 .mu.L of Borate
buffer (500 mM sodium borate/500 mM sodium chloride, pH 8.0)
followed by 300 .mu.L of Dithiothreitol (DTT, 100 mM solution in
H.sub.2O). The reaction mixture is stirred using a vortex
instrument and incubated at 37.degree. C. for 30 min. Three PD10
columns are equilibrated with PBS containing 1 mM DTPA (in PBS) and
the reduced antibody is eluted through the three PD10 columns and
collected in 4.2 mL PBS/DTPA solution (1.4 mL per column). The
reduced antibody is then stored on ice. The number of thiols per
antibody and the antibody concentration are determined according to
General Procedure N.
General Procedure N: Determination of Number of Thiols Per
Ligand
A reference sample of a Ligand or a sample of an antibody reduced
according to General Procedure L is diluted to about 1:40 (w/w) in
PBS, and the uv absorbance of the solution is measured at 280 nm
using standard uv spectroscopic methods. Preferably, the ratio of
Ligand:PBS in the solution is such that the uv absorbance ranges
from about 0.13-0.2 AU (absorbance units).
A test sample of a Ligand or a test sample of an antibody reduced
according to General Procedure L is diluted to about 1:20 with a
PBS solution containing about 15 .mu.L DTNB stock solution/mL PBS.
A blank sample containing DTNB at the same concentration as the
test solution (i.e., 15 .mu.L DTNB stock/mL PBS) is then prepared.
The spectrophotometer is referenced at zero nm with the blank
sample, then the absorbance of the test sample is measured at 412
nm.
The molar concentration of the antibody is then determined using
the formula: [Ligand]=(OD.sub.280/2.24e.sup.5).times.dilution
factor.
The molar concentration of thiol is then determined using the
formula: [--SH]=(OD.sub.412/1.415e.sup.4).times.dilution
factor.
The [SH]/[Ligand] ratio is then calculated. A reduced monoclonal
antibody Ligand can have from 1 to about 20 sulfhydryl groups, but
typically has between about 6 to about 9 sulfhydryl groups. In a
preferred embodiment, the [SH]/[Ligand] ratio range is from about 7
to about 9.
It is understood that the [SH]/[Ligand] ratio is the average number
of -A.sub.a-W.sub.w-Y.sub.y-D units per Ligand unit.
General Procedure O: Determination of the number of Drug molecules
per Antibody in a Drug-Linker-Antibody Conjugate. The Drug:Antibody
ratio for a Drug-Linker-Antibody Conjugate is determined by
measuring the number of Dithiothreitol (DTT) reducible thiols that
remain after conjugation, using the following method: A 200 mL
sample of a Drug-Linker-Antibody conjugate is treated with DTT (100
mM solution in water) to bring the concentration to 10 mM DTT. The
resulting solution is incubated at 37.degree. C. for 30 min, then
eluted through a PD10 column using PBS/DTPA as the eluent. The
OD.sub.280 of the reduced conjugate is then measured and the molar
concentration is measured according to General Procedure Q.
The molar concentration of thiol is determined using DTNB as
described in General Procedure M. The ratio of thiol concentration
to antibody concentration is then calculated and the Drug:Ligand
ratio is the difference between the Thiol:Antibody ratio
(determined using General Procedure N) and the Drug:Antibody ratio
as determined in the previous paragraph.
General Procedure P: Determination of the amount of quenched
Drug-Linker compound in a Drug-Linker-Antibody Conjugate. This
assay provides a quantitative determination of the Drug-Linker in
the Drug-Linker-Antibody conjugate that is not covalently bound to
Antibody. Assuming that all maleimide groups of Drug-Linker in the
reaction mixture have been quenched with Cysteine, the unbound drug
is the Cysteine quenched adduct of the Drug-Linker Compound, i.e.
Drug-Linker-Cys. The proteinaceous Drug-Linker-Antibody Conjugate
is denatured, precipitated, and isolated by centrifugation under
conditions in which the Drug-Linker-Cys is soluble. The unbound
Drug-Linker-Cys is detected quantitatively by HPLC, and the
resulting chromatogram is compared to a standard curve to determine
the concentration of unbound Drug-Linker-Cys in the sample. This
concentration is divided by the total concentration of Drug in the
conjugate as determined using General Procedure 0 and General
Procedure Q.
Specifically, 100 mL of a 100 .mu.M Drug-Linker-Cys adduct "working
solution" is prepared by adding 1 .mu.L of 100 mM Cysteine in
PBS/DTPA and an appropriate volume of stock solution of a
Drug-Linker compound to 98 .mu.L of 50% methanol/PBS. The
"appropriate volume" in liters is calculated using the formula:
V=1c-8/[Drug-Linker]. Six tubes are then labelled as follows: "0",
"0.5", "1", "2", "3", and "5", and appropriate amounts of working
solution are placed in each tube and diluted with 50% methanol/PBS
to give a total volume of 100 mL in each tube. The labels indicate
the .mu.M concentration of the standards.
A 50 .mu.L solution of a Drug-Linker-Antibody Conjugate and a 50
.mu.L solution of the Cysteine quenched reaction mixture ("qrm")
are collected in separate test tubes and are each diluted with 50
.mu.L, of methanol that has been cooled to -20.degree. C. The
samples are then cooled to -20.degree. C. over 10 min.
The samples are then centrifuged at 13000 rpm in a desktop
centrifuge for 10 min. The supernatants are transferred to HPLC
vials, and 90 .mu.L aliquots of each sample are separately analyzed
using HPLC (C12 RP column (Phenomenex); monitored at the absorbance
maximum of the Drug-Linker Compound using a flow rate of 1.0
mL/min. The eluent used is a linear gradient of MeCN ranging from
10 to 90% in aqueous 5 mM ammonium phosphate, pH 7.4, over 10 min;
then 90% MeCN over 5 min.; then returning to initial conditions).
The Drug-Linker-Cys adduct typically elutes between about 7 and
about 10 minutes.
A standard curve is then prepared by plotting the Peak Area of the
standards vs. their concentration (in .mu.M). Linear regression
analysis is performed to determine the equation and correlation
coefficient of the standard curve. R2 values are typically
>0.99. From the regression equation is determined the
concentration of the Drug-Linker-Cys adduct in the HPLC sample and
in the conjugate, using the formulas: [Drug-Linker-Cys].sub.(HPLC
spl)=(Peak area-intercept)/slope;
[Drug-Linker-Cys].sub.(conjugate)=2.times.[Drug-Linker-Cys].sub.(HPLC
spl)
The percent of Drug-Linker-Cys adduct present can be determined
using the formula: %
Drug-Linker-Cys=100.times.[Drug-Linker-Cys].sub.(conjugate)/[drug]
where [drug]=[Conjugate].times.drug/Ab, [Conjugate] is determined
using the conjugate concentration assay, and the Drug: Antibody
ratio is determined using the Drug: Antibody ratio assay.
General Procedure Q: Determination of Drug-Linker-Antibody
Conjugate concentration for drug linkers with minimal uv absorbance
at 280 nm. The concentration of Drug-Linker-Antibody conjugate can
be determined in the same manner for the concentration of the
parent antibody, by measuring the absorbance at 280 nm of an
appropriate dilution, using the following formula:
[Conjugate](mg/mL)=(OD.sub.280.times.dilution
factor/1.4).times.0.9
Determination of Drug-Linker-Antibody Conjugate concentration for
drug linkers with substantial uv absorbance at 280 nm (e.g.
Compounds 68 and 69). Because the absorbances of Compounds 68 and
69 overlap with the absorbances of an antibody, spectrophotometric
determination of the conjugate concentration is most useful when
the measurement is performed using the absorbances at both 270 nm
and 280 nm. Using this data, the molar concentration of
Drug-Linker-Ligand conjugate is given by the following formula:
[Conjugate]=(OD.sub.280.times.1.23
e.sup.-5-OD.sub.270.times.9.35e.sup.-6).times.dilution factor where
the 1.23e.sup.-5 and 9.35e.sup.-6 are calculated from the molar
extinction coefficients of the drug and the antibody, which are
estimated as: e.sub.270 Drug=2.06e.sup.4 e.sub.270
Antibody=1.87e.sup.5 e.sub.280 Drug=1.57e.sup.4 e.sub.280
Antibody=2.24e.sup.5
Determination of Drug-Linker-Antibody Conjugate concentration for
drug linkers with substantial uv absorbance at 280 nm (e.g.
Compounds 68 and 69). Because the absorbances of Compounds 68 and
69 overlap with the absorbances of an antibody, spectrophotometric
determination of the conjugate concentration is most useful when
the measurement is performed using the absorbances at both 270 nm
and 280 nm. Using this data, the molar concentration of
Drug-Linker-Ligand conjugate is given by the following formula:
[Conjugate]=(OD.sub.280.times.1.23
e.sup.-5-OD.sub.270.times.9.35e.sup.-6).times.dilution factor where
the values 1.23e.sup.-5 and 9.35e.sup.-6 are calculated from the
molar extinction coefficients of the drug and the antibody, which
are estimated as: e.sub.270 Drug=2.06e.sup.4 e.sub.270
Antibody=1.87e.sup.5 e.sub.280 Drug=1.57e.sup.4 e.sub.280
Antibody=2.24e.sup.5
General Procedure R: Determination of the aggregation state of The
Antibody in a Drug-Linker-Antibody Conjugate. A suitable quantity
(-10 .mu.g) of a Drug-Linker-Antibody Conjugate is eluted through a
size-exclusion chromatography (SEC) column (Tosoh Biosep SW3000 4.6
mm.times.30 cm eluted at 0.35 mL/min. with PBS) under standard
conditions. Chromatograms are obtained at 220 nm and 280 nm and the
OD.sub.280/OD.sub.220 ratio is calculated. The corresponding
aggregate typically has a retention time of between about 5.5 and
about 7 min, and has about the same OD.sub.280/OD.sub.220 ratio as
the monomeric Drug-Linker-Antibody Conjugate.
Compositions
In other aspects, the present invention provides a composition
comprising an effective amount of a Compound of the Invention and a
pharmaceutically acceptable carrier or vehicle. For convenience,
the Drug units, Drug-Linker Compounds and Drug-Linker-Ligand
Conjugates of the invention can simply be referred to as compounds
of the invention. The compositions are suitable for veterinary or
human administration.
The compositions of the present invention can be in any form that
allows for the composition to be administered to an animal. For
example, the composition can be in the form of a solid, liquid or
gas (aerosol). Typical routes of administration include, without
limitation, oral, topical, parenteral, sublingual, rectal, vaginal,
ocular, and intranasal. Parenteral administration includes
subcutaneous injections, intravenous, intramuscular, intrasternal
injection or infusion techniques. Preferably, the compositions are
administered parenterally. Pharmaceutical compositions of the
invention can be formulated so as to allow a Compound of the
Invention to be bioavailable upon administration of the composition
to an animal. Compositions can take the form of one or more dosage
units, where for example, a tablet can be a single dosage unit, and
a container of a Compound of the Invention in aerosol form can hold
a plurality of dosage units.
Materials used in preparing the pharmaceutical compositions can be
non-toxic in the amounts used. It will be evident to those of
ordinary skill in the art that the optimal dosage of the active
ingredient(s) in the pharmaceutical composition will depend on a
variety of factors. Relevant factors include, without limitation,
the type of animal (e.g., human), the particular form of the
Compound of the Invention, the manner of administration, and the
composition employed.
The pharmaceutically acceptable carrier or vehicle can be
particulate, so that the compositions are, for example, in tablet
or powder form. The carrier(s) can be liquid, with the compositions
being, for example, an oral syrup or injectable liquid. In
addition, the carrier(s) can be gaseous, so as to provide an
aerosol composition useful in, e.g., inhalatory administration.
When intended for oral administration, the composition is
preferably in solid or liquid form, where semi-solid, semi-liquid,
suspension and gel forms are included within the forms considered
herein as either solid or liquid.
As a solid composition for oral administration, the composition can
be formulated into a powder, granule, compressed tablet, pill,
capsule, chewing gum, wafer or the like form. Such a solid
composition typically contains one or more inert diluents. In
addition, one or more of the following can be present: binders such
as carboxymethylcellulose, ethyl cellulose, microcrystalline
cellulose, or gelatin; excipients such as starch, lactose or
dextrins, disintegrating agents such as alginic acid, sodium
alginate, Primogel, corn starch and the like; lubricants such as
magnesium stearate or Sterotex; glidants such as colloidal silicon
dioxide; sweetening agents such as sucrose or saccharin, a
flavoring agent such as peppermint, methyl salicylate or orange
flavoring, and a coloring agent.
When the composition is in the form of a capsule, e.g., a gelatin
capsule, it can contain, in addition to materials of the above
type, a liquid carrier such as polyethylene glycol, cyclodextrin or
a fatty oil.
The composition can be in the form of a liquid, e.g., an elixir,
syrup, solution, emulsion or suspension. The liquid can be useful
for oral administration or for delivery by injection. When intended
for oral administration, a composition can comprise one or more of
a sweetening agent, preservatives, dye/colorant and flavor
enhancer. In a composition for administration by injection, one or
more of a surfactant, preservative, wetting agent, dispersing
agent, suspending agent, buffer, stabilizer and isotonic agent can
also be included.
The liquid compositions of the invention, whether they are
solutions, suspensions or other like form, can also include one or
more of the following: sterile diluents such as water for
injection, saline solution, preferably physiological saline,
Ringer's solution, isotonic sodium chloride, fixed oils such as
synthetic mono or digylcerides which can serve as the solvent or
suspending medium, polyethylene glycols, glycerin, cyclodextrin,
propylene glycol or other solvents; antibacterial agents such as
benzyl alcohol or methyl paraben; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. A parenteral composition can be
enclosed in ampoule, a disposable syringe or a multiple-dose vial
made of glass, plastic or other material. Physiological saline is a
preferred adjuvant. An injectable composition is preferably
sterile.
The amount of the Compound of the Invention that is effective in
the treatment of a particular disorder or condition will depend on
the nature of the disorder or condition, and can be determined by
standard clinical techniques. In addition, in vitro or in vivo
assays can optionally be employed to help identify optimal dosage
ranges. The precise dose to be employed in the compositions will
also depend on the route of administration, and the seriousness of
the disease or disorder, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
The compositions comprise an effective amount of a Compound of the
Invention such that a suitable dosage will be obtained. Typically,
this amount is at least about 0.01% of a Compound of the Invention
by weight of the composition. When intended for oral
administration, this amount can be varied to range from about 0.1%
to about 80% by weight of the composition. Preferred oral
compositions can comprise from about 4% to about 50% of the
Compound of the Invention by weight of the composition. Preferred
compositions of the present invention are prepared so that a
parenteral dosage unit contains from about 0.01% to about 2% by
weight of the Compound of the Invention.
For intravenous administration, the composition can comprise from
about 1 to about 250 mg of a Compound of the Invention per kg of
the animal's body weight. Preferably, the amount administered will
be in the range from about 4 to about 25 mg/kg of body weight of
the Compound of the Invention.
Generally, the dosage of Compound of the Invention administered to
an animal is typically about 0.1 mg/kg to about 250 mg/kg of the
animal's body weight. Preferably, the dosage administered to an
animal is between about 0.1 mg/kg and about 20 mg/kg of the
animal's body weight, more preferably about 1 mg/kg to about 10
mg/kg of the animal's body weight.
The Compounds of the Invention or compositions can be administered
by any convenient route, for example by infusion or bolus
injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.).
Administration can be systemic or local. Various delivery systems
are known, e.g., encapsulation in liposomes, microparticles,
microcapsules, capsules, etc., and can be used to administer a
Compound of the Invention or composition. In certain embodiments,
more than one Compound of the Invention or composition is
administered to an animal. Methods of administration include, but
are not limited to, oral administration and parenteral
administration; parenteral administration including, but not
limited to, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous; intranasal, epidural, sublingual,
intranasal, intracerebral, intraventricular, intrathecal,
intravaginal, transdermal, rectally, by inhalation, or topically to
the ears, nose, eyes, or skin. The preferred mode of administration
is left to the discretion of the practitioner, and will depend
in-part upon the site of the medical condition (such as the site of
cancer or autoimmune disease).
In a preferred embodiment, the present Compounds of the Invention
or compositions are administered parenterally.
In a more preferred embodiment, the present Compounds of the
Invention or compositions are administered intravenously.
In specific embodiments, it can be desirable to administer one or
more Compounds of the Invention or compositions locally to the area
in need of treatment. This can be achieved, for example, and not by
way of limitation, by local infusion during surgery; topical
application, e.g., in conjunction with a wound dressing after
surgery; by injection; by means of a catheter; by means of a
suppository; or by means of an implant, the implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. In one embodiment,
administration can be by direct injection at the site (or former
site) of a cancer, tumor or neoplastic or pre-neoplastic tissue. In
another embodiment, administration can be by direct injection at
the site (or former site) of a manifestation of an autoimmune
disease.
In certain embodiments, it can be desirable to introduce one or
more Compounds of the Invention or compositions into the central
nervous system by any suitable route, including intraventricular
and intrathecal injection. Intraventricular injection can be
facilitated by an intraventricular catheter, for example, attached
to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing agent,
or via perfusion in a fluorocarbon or synthetic pulmonary
surfactant. In certain embodiments, the Compounds of the Invention
or compositions can be formulated as a suppository, with
traditional binders and carriers such as triglycerides.
In another embodiment, the Compounds of the invention can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein,
ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the Compounds of the Invention or
compositions can be delivered in a controlled release system. In
one embodiment, a pump can be used (see Langer, supra; Sefton, CRC
Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery
88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In
another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,
New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev.
Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190
(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al.,
J. Neurosurg. 71:105 (1989)). In yet another embodiment, a
controlled-release system can be placed in proximity of the target
of the Compounds of the Invention or compositions, e.g., the brain,
thus requiring only a fraction of the systemic dose (see, e.g.,
Goodson, in Medical Applications of Controlled Release, supra, vol.
2, pp. 115-138 (1984)). Other controlled-release systems discussed
in the review by Langer (Science 249:1527-1533 (1990)) can be
used.
The term "carrier" refers to a diluent, adjuvant or excipient, with
which a Compound of the Invention is administered. Such
pharmaceutical carriers can be liquids, such as water and oils,
including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. The carriers can be saline, gum acacia, gelatin,
starch paste, talc, keratin, colloidal silica, urea, and the like.
In addition, auxiliary, stabilizing, thickening, lubricating and
coloring agents can be used. In one embodiment, when administered
to an animal, the Compounds of the Invention or compositions and
pharmaceutically acceptable carriers are sterile. Water is a
preferred carrier when the Compounds of the Invention are
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
carriers also include excipients such as starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
present compositions, if desired, can also contain minor amounts of
wetting or emulsifying agents, or pH buffering agents.
The present compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, pellets, capsules, capsules
containing liquids, powders, sustained-release formulations,
suppositories, emulsions, aerosols, sprays, suspensions, or any
other form suitable for use. In one embodiment, the
pharmaceutically acceptable carrier is a capsule (see e.g., U.S.
Pat. No. 5,698,155). Other examples of suitable pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by
E. W. Martin.
In a preferred embodiment, the Compounds of the Invention are
formulated in accordance with routine procedures as a
pharmaceutical composition adapted for intravenous administration
to animals, particularly human beings. Typically, the carriers or
vehicles for intravenous administration are sterile isotonic
aqueous buffer solutions. Where necessary, the compositions can
also include a solubilizing agent. Compositions for intravenous
administration can optionally comprise a local anesthetic such as
lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
a Compound of the Invention is to be administered by infusion, it
can be dispensed, for example, with an infusion bottle containing
sterile pharmaceutical grade water or saline. Where the Compound of
the Invention is administered by injection, an ampoule of sterile
water for injection or saline can be provided so that the
ingredients can be mixed prior to administration.
Compositions for oral delivery can be in the form of tablets,
lozenges, aqueous or oily suspensions, granules, powders,
emulsions, capsules, syrups, or elixirs, for example. Orally
administered compositions can contain one or more optionally
agents, for example, sweetening agents such as fructose, aspartame
or saccharin; flavoring agents such as peppermint, oil of
wintergreen, or cherry; coloring agents; and preserving agents, to
provide a pharmaceutically palatable preparation. Moreover, where
in tablet or pill form, the compositions can be coated to delay
disintegration and absorption in the gastrointestinal tract thereby
providing a sustained action over an extended period of time.
Selectively permeable membranes surrounding an osmotically active
driving compound are also suitable for orally administered
compounds. In these later platforms, fluid from the environment
surrounding the capsule is imbibed by the driving compound, which
swells to displace the agent or agent composition through an
aperture. These delivery platforms can provide an essentially zero
order delivery profile as opposed to the spiked profiles of
immediate release formulations. A time-delay material such as
glycerol monostearate or glycerol stearate can also be used. Oral
compositions can include standard carriers such as mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate, etc. Such carriers are preferably of
pharmaceutical grade.
The compositions can be intended for topical administration, in
which case the carrier may be in the form of a solution, emulsion,
ointment or gel base. The base, for example, can comprise one or
more of the following: petrolatum, lanolin, polyethylene glycols,
beeswax, mineral oil, diluents such as water and alcohol, and
emulsifiers and stabilizers. Thickening agents can be present in a
composition for topical administration. If intended for transdermal
administration, the composition can be in the form of a transdermal
patch or an iontophoresis device. Topical formulations can comprise
a concentration of a Compound of the Invention of from about 0.1%
to about 10% w/v (weight per unit volume of composition).
The composition can be intended for rectal administration, in the
form, e.g., of a suppository which will melt in the rectum and
release the Compound of the Invention. The composition for rectal
administration can contain an oleaginous base as a suitable
nonirritating excipient. Such bases include, without limitation,
lanolin, cocoa butter and polyethylene glycol.
The composition can include various materials that modify the
physical form of a solid or liquid dosage unit. For example, the
composition can include materials that form a coating shell around
the active ingredients. The materials that form the coating shell
are typically inert, and can be selected from, for example, sugar,
shellac, and other enteric coating agents. Alternatively, the
active ingredients can be encased in a gelatin capsule.
The compositions can consist of gaseous dosage units, e.g., it can
be in the form of an aerosol. The term aerosol is used to denote a
variety of systems ranging from those of colloidal nature to
systems consisting of pressurized packages. Delivery can be by a
liquefied or compressed gas or by a suitable pump system that
dispenses the active ingredients. Aerosols of Compounds of the
Invention can be delivered in single phase, bi-phasic, or
tri-phasic systems in order to deliver the Compound(s) of the
Invention. Delivery of the aerosol includes the necessary
container, activators, valves, subcontainers, Spacers and the like,
which together can form a kit. Preferred aerosols can be determined
by one skilled in the art, without undue experimentation.
Whether in solid, liquid or gaseous form, the compositions of the
present invention can comprise a pharmacological agent used in the
treatment of cancer, an autoimmune disease or an infectious
disease.
The pharmaceutical compositions can be prepared using methodology
well known in the pharmaceutical art. For example, a composition
intended to be administered by injection can be prepared by
combining a Compound of the Invention with water so as to form a
solution. A surfactant can be added to facilitate the formation of
a homogeneous solution or suspension. Surfactants are compounds
that non-covalently interact with a Compound of the Invention so as
to facilitate dissolution or homogeneous suspension of the active
compound in the aqueous delivery system.
Therapeutic Uses of the Compounds of the Invention
The Compounds of the Invention are useful for treating cancer, an
autoimmune disease or an infectious disease in an animal.
Treatment of Cancer
The Compounds of the Invention are useful for inhibiting the
multiplication of a tumor cell or cancer cell, or for treating
cancer in an animal. The Compounds of the Invention can be used
accordingly in a variety of settings for the treatment of animal
cancers. The Drug-Linker-Ligand Conjugates can be used to deliver a
Drug or Drug unit to a tumor cell or cancer cell. Without being
bound by theory, in one embodiment, the Ligand unit of a Compound
of the Invention binds to or associates with a cancer-cell or a
tumor-cell-associated antigen, and the Compound of the Invention
can be taken up inside a tumor cell or cancer cell through
receptor-mediated endocytosis. The antigen can be attached to a
tumor cell or cancer cell or can be an extracellular matrix protein
associated with the tumor cell or cancer cell. Once inside the
cell, one or more specific peptide sequences within the Linker unit
are hydrolytically cleaved by one or more tumor-cell or
cancer-cell-associated proteases, resulting in release of a Drug or
a Drug-Linker Compound. The released Drug or Drug-Linker Compound
is then free to migrate in the cytosol and induce cytotoxic
activities. In an alternative embodiment, the Drug or Drug unit is
cleaved from the Compound of the Invention outside the tumor cell
or cancer cell, and the Drug or Drug-Linker Compound subsequently
penetrates the cell.
In one embodiment, the Ligand unit binds to the tumor cell or
cancer cell.
In another embodiment, the Ligand unit binds to a tumor cell or
cancer cell antigen which is on the surface of the tumor cell or
cancer cell.
In another embodiment, the Ligand unit binds to a tumor cell or
cancer cell antigen which is an extracellular matrix protein
associated with the tumor cell or cancer cell.
In one embodiment, the tumor cell or cancer cell is of the type of
tumor or cancer that the animal needs treatment or prevention
of.
The specificity of the Ligand unit for a particular tumor cell or
cancer cell can be important for determining those tumors or
cancers that are most effectively treated. For example, Compounds
of the Invention having a BR96 Ligand unit can be useful for
treating antigen positive carcinomas including those of the lung,
breast, colon, ovaries, and pancreas. Compounds of the Invention
having an Anti-CD30 or an anti-CD40 Ligand unit can be useful for
treating hematologic malignancies.
Other particular types of cancers that can be treated with
Compounds of the Invention include, but are not limited to, those
disclosed in Table 3.
TABLE-US-00006 TABLE 3 Solid tumors, including but not limited to:
fibrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic
sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma
lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor
leiomyosarcoma rhabdomyosarcoma colon cancer colorectal cancer
kidney cancer pancreatic cancer bone cancer breast cancer ovarian
cancer prostate cancer esophogeal cancer stomach cancer oral cancer
nasal cancer throat cancer squamous cell carcinoma basal cell
carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland
carcinoma papillary carcinoma papillary adenocarcinomas
cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal
cell carcinoma hepatoma bile duct carcinoma choriocarcinoma
seminoma embryonal carcinoma Wilms' tumor cervical cancer uterine
cancer testicular cancer small cell lung carcinoma bladder
carcinoma lung cancer epithelial carcinoma glioma glioblastoma
multiforme astrocytoma medulloblastoma craniopharyngioma ependymoma
pinealoma hemangioblastoma acoustic neuroma oligodendroglioma
meningioma skin cancer melanoma neuroblastoma retinoblastoma
blood-borne cancers, including but not limited to: acute
lymphoblastic leukemia "ALL" acute lymphoblastic B-cell leukemia
acute lymphoblastic T-cell leukemia acute myeloblastic leukemia
"AML" acute promyelocytic leukemia "APL" acute monoblastic leukemia
acute erythroleukemic leukemia acute megakaryoblastic leukemia
acute myelomonocytic leukemia acute nonlymphocyctic leukemia acute
undifferentiated leukemia chronic myelocytic leukemia "CML" chronic
lymphocytic leukemia "CLL" hairy cell leukemia multiple myeloma
acute and chronic leukemias: lymphoblastic myelogenous lymphocytic
myelocytic leukemias Lymphomas: Hodgkin's disease non-Hodgkin's
Lymphoma Multiple myeloma Waldenstrom's macroglobulinemia Heavy
chain disease Polycythemia vera
The Compounds of the Invention can also be used as
chemotherapeutics in the untargeted form. For example, the Drugs
themselves, or the Drug-Linker Compounds are useful for treating
ovarian, CNS, renal, lung, colon, melanoma, or hematologic cancers
or tumors.
The Compounds of the Invention provide Conjugation specific tumor
or cancer targeting, thus reducing general toxicity of these
compounds. The Linker units stabilize the Compounds of the
Invention in blood, yet are cleavable by tumor-specific proteases
within the cell, liberating a Drug.
Multi-Modality Therapy for Cancer
Cancer, including, but not limited to, a tumor, metastasis, or any
disease or disorder characterized by uncontrolled cell growth, can
be treated or prevented by administration of a Compound of the
Invention.
In other embodiments, the invention provides methods for treating
or preventing cancer, comprising administering to an animal in need
thereof an effective amount of a Compound of the Invention and a
chemotherapeutic agent. In one embodiment the chemotherapeutic
agent is that with which treatment of the cancer has not been found
to be refractory. In another embodiment, the chemotherapeutic agent
is that with which the treatment of cancer has been found to be
refractory. The Compounds of the Invention can be administered to
an animal that has also undergone surgery as treatment for the
cancer.
In one embodiment, the additional method of treatment is radiation
therapy.
In a specific embodiment, the Compound of the Invention is
administered concurrently with the chemotherapeutic agent or with
radiation therapy. In another specific embodiment, the
chemotherapeutic agent or radiation therapy is administered prior
or subsequent to administration of a Compound of the Invention,
preferably at least an hour, five hours, 12 hours, a day, a week, a
month, more preferably several months (e.g., up to three months),
prior or subsequent to administration of a Compound of the
Invention.
A chemotherapeutic agent can be administered over a series of
sessions, any one or a combination of the chemotherapeutic agents
listed in Table 4 can be administered. With respect to radiation,
any radiation therapy protocol can be used depending upon the type
of cancer to be treated. For example, but not by way of limitation,
x-ray radiation can be administered; in particular, high-energy
megavoltage (radiation of greater that 1 MeV energy) can be used
for deep tumors, and electron beam and orthovoltage x-ray radiation
can be used for skin cancers. Gamma-ray emitting radioisotopes,
such as radioactive isotopes of radium, cobalt and other elements,
can also be administered.
Additionally, the invention provides methods of treatment of cancer
with a Compound of the Invention as an alternative to chemotherapy
or radiation therapy where the chemotherapy or the radiation
therapy has proven or can prove too toxic, e.g., results in
unacceptable or unbearable side effects, for the subject being
treated. The animal being treated can, optionally, be treated with
another cancer treatment such as surgery, radiation therapy or
chemotherapy, depending on which treatment is found to be
acceptable or bearable.
The Compounds of the Invention can also be used in an in vitro or
ex vivo fashion, such as for the treatment of certain cancers,
including, but not limited to leukemias and lymphomas, such
treatment involving autologous stem cell transplants. This can
involve a multi-step process in which the animal's autologous
hematopoietic stem cells are harvested and purged of all cancer
cells, the patient's remaining bone-marrow cell population is then
eradicated via the administration of a high dose of a Compound of
the Invention with or without accompanying high dose radiation
therapy, and the stem cell graft is infused back into the animal.
Supportive care is then provided while bone marrow function is
restored and the animal recovers.
Multi-Drug Therapy for Cancer
The present invention includes methods for treating cancer,
comprising administering to an animal in need thereof an effective
amount of a Compound of the Invention and another therapeutic agent
that is an anti-cancer agent. Suitable anticancer agents include,
but are not limited to, methotrexate, taxol, L-asparaginase,
mercaptopurine, thioguanine, hydroxyurea, cytarabine,
cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,
mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards,
cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan,
camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin,
dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,
vincristine, vinorelbine, paclitaxel, and docetaxel. In a preferred
embodiment, the anti-cancer agent includes, but is not limited to,
a drug listed in Table 4.
TABLE-US-00007 TABLE 4 Alkylating agents Nitrogen mustards:
cyclophosphamide Ifosfamide trofosfamide Chlorambucil Nitrosoureas:
carmustine (BCNU) Lomustine (CCNU) Alkylsulphonates busulfan
Treosulfan Triazenes: Dacarbazine Platinum containing compounds:
Cisplatin carboplatin Plant Alkaloids Vinca alkaloids: vincristine
Vinblastine Vindesine Vinorelbine Taxoids: paclitaxel Docetaxol DNA
Topoisomerase Inhibitors Epipodophyllins: etoposide Teniposide
Topotecan 9-aminocamptothecin camptothecin crisnatol mitomycins:
Mitomycin C Anti-metabolites Anti-folates: DHFR inhibitors:
methotrexate Trimetrexate IMP dehydrogenase Inhibitors:
mycophenolic acid Tiazofurin Ribavirin EICAR Ribonuclotide
reductase Inhibitors: hydroxyurea deferoxamine Pyrimidine analogs:
Uracil analogs 5-Fluorouracil Floxuridine Doxifluridine Ratitrexed
Cytosine analogs cytarabine (ara C) Cytosine arabinoside
fludarabine Purine analogs: mercaptopurine Thioguanine Hormonal
therapies: Receptor antagonists: Anti-estrogen Tamoxifen Raloxifene
megestrol LHRH agonists: goscrclin Leuprolide acetate
Anti-androgens: flutamide bicalutamide Retinoids/Deltoids Vitamin
D3 analogs: EB 1089 CB 1093 KH 1060 Photodynamic therapies:
vertoporfin (BPD-MA) Phthalocyanine photosensitizer Pc4
Demethoxy-hypocrellin A (2BA-2-DMHA) Cytokines: Interferon-.alpha.
Interferon-.gamma. Tumor necrosis factor Others: Isoprenylation
inhibitors: Lovastatin Dopaminergic neurotoxins:
1-methyl-4-phenylpyridinium ion Cell cycle inhibitors:
staurosporine Actinomycins: Actinomycin D Dactinomycin Bleomycins:
bleomycin A2 Bleomycin B2 Peplomycin Anthracyclines: daunorubicin
Doxorubicin (adriamycin) Idarubicin Epirubicin Pirarubicin
Zorubicin Mitoxantrone MDR inhibitors: verapamil Ca.sup.2+ATPase
inhibitors: thapsigargin
Treatment of Autoimmune Diseases
The Compounds of the Invention are useful for killing or inhibiting
the replication of a cell that produces an autoimmune disease or
for treating an autoimmune disease. The Compounds of the Invention
can be used accordingly in a variety of settings for the treatment
of an autoimmune disease in an animal. The Drug-Linker-Ligand
Conjugates can be used to deliver a Drug to a target cell. Without
being bound by theory, in one embodiment, the Drug-Linker-Ligand
Conjugate associates with an antigen on the surface of a target
cell, and the Compound of the Invention is then taken up inside a
target-cell through receptor-mediated endocytosis. Once inside the
cell, one or more specific peptide sequences within the Linker unit
are enzymatically or hydrolytically cleaved, resulting in release
of a Drug. The released Drug is then free to migrate in the cytosol
and induce cytotoxic activities. In an alternative embodiment, the
Drug is cleaved from the Compound of the Invention outside the
target cell, and the Drug subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to an autoimmune
antigen.
In another embodiment, the Ligand unit binds to an autoimmune
antigen which is on the surface of a cell.
In another embodiment, the target cell is of the type of cell that
produces the autoimmune antigen which causes the disease the animal
needs treatment or prevention of.
In a preferred embodiment, the Ligand binds to activated lympocytes
that are associated with the autoimmune diesease state.
In a further embodiment, the Compounds of the Invention kill or
inhibit the multiplication of cells that produce an auto-immune
antibody associated with a particular autoimmune disease.
Particular types of autoimmune diseases that can be treated with
the Compounds of the Invention include, but are not limited to,
Th2-lymphocyte related disorders (e.g., atopic dermatitis, atopic
asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome,
systemic sclerosis, and graft versus host disease); Th1
lymphocyte-related disorders (e.g., rheumatoid arthritis, multiple
sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis,
Grave's disease, primary biliary cirrhosis, Wegener's
granulomatosis, and tuberculosis); activated B lymphocyte-related
disorders (e.g., systemic lupus erythematosus, Goodpasture's
syndrome, rheumatoid arthritis, and type I diabetes); and those
disclosed in Table 5.
TABLE-US-00008 TABLE 5 Active Chronic Hepatitis Addison's Disease
Allergic Alveolitis Allergic Reaction Allergic Rhinitis Alport's
Syndrome Anaphlaxis Ankylosing Spondylitis Anti-phosholipid
Syndrome Arthritis Ascariasis Aspergillosis Atopic Allergy Atropic
Dermatitis Atropic Rhinitis Behcet's Disease Bird-Fancier's Lung
Bronchial Asthma Caplan's Syndrome Cardiomyopathy Celiac Disease
Chagas' Disease Chronic Glomerulonephritis Cogan's Syndrome Cold
Agglutinin Disease Congenital Rubella Infection CREST Syndrome
Crohn's Disease Cryoglobulinemia Cushing's Syndrome Dermatomyositis
Discoid Lupus Dressler's Syndrome Eaton-Lambert Syndrome Echovirus
Infection Encephalomyelitis Endocrine opthalmopathy Epstein-Barr
Virus Infection Equine Heaves Erythematosis Evan's Syndrome Felty's
Syndrome Fibromyalgia Fuch's Cyclitis Gastric Atrophy
Gastrointestinal Allergy Giant Cell Arteritis Glomerulonephritis
Goodpasture's Syndrome Graft v. Host Disease Graves' Disease
Guillain-Barre Disease Hashimoto's Thyroiditis Hemolytic Anemia
Henoch-Schonlein Purpura Idiopathic Adrenal Atrophy Idiopathic
Pulmonary Fibritis IgA Nephropathy Inflammatory Bowel Diseases
Insulin-dependent Diabetes Mellitus Juvenile Arthritis Juvenile
Diabetes Mellitus (Type I) Lambert-Eaton Syndrome Laminitis Lichen
Planus Lupoid Hepatitis Lupus Lymphopenia Meniere's Disease Mixed
Connective Tissue Disease Multiple Sclerosis Myasthenia Gravis
Pernicious Anemia Polyglandular Syndromes Presenile Dementia
Primary Agammaglobulinemia Primary Biliary Cirrhosis Psoriasis
Psoriatic Arthritis Raynauds Phenomenon Recurrent Abortion Reiter's
Syndrome Rheumatic Fever Rheumatoid Arthritis Sampter's Syndrome
Schistosomiasis Schmidt's Syndrome Scleroderma Shulman's Syndrome
Sjorgen's Syndrome Stiff-Man Syndrome Sympathetic Ophthalmia
Systemic Lupus Erythematosis Takayasu's Arteritis Temporal
Arteritis Thyroiditis Thrombocytopenia Thyrotoxicosis Toxic
Epidermal Necrolysis Type B Insulin Resistance Type I Diabetes
Mellitus Ulcerative Colitis Uveitis Vitiligo Waldenstrom's
Macroglobulemia Wegener's Granulomatosis
Multi-Drug Therapy of Autoimmune Diseases
The present invention also provides methods for treating an
autoimmune disease, comprising administering to an animal in need
thereof an effective amount of a Compound of the Invention and
another therapeutic agent that known for the treatment of an
autoimmune disease. In one embodiment, the anti-autoimmune disease
agent includes, but is not limited to, agents listed in Table
6.
TABLE-US-00009 TABLE 6 cyclosporine cyclosporine A mycophenylate
mofetil sirolimus tacrolimus enanercept prednisone azathioprine
methotrexate cyclophosphamide prednisone aminocaproic acid
chloroquine hydroxychloroquine hydrocortisone dexamethasone
chlorambucil DHEA danazol bromocriptine meloxicam infliximab
Treatment of Infectious Diseases
The Compounds of the Invention are useful for killing or inhibiting
the multiplication of a cell that produces an infectious disease or
for treating an infectious disease. The Compounds of the Invention
can be used accordingly in a variety of settings for the treatment
of an infectious disease in an animal. The Drug-Linker-Ligand
Conjugates can be used to deliver a Drug to a target cell. Without
being bound by theory, in one embodiment, the Drug-Linker-Ligand
Conjugate associates with an antigen on the surface of a target
cell, and the Compound of the Invention is then taken up inside a
target-cell through receptor-mediated endocytosis. Once inside the
cell, one or more specific peptide sequences within the Linker unit
are enzymatically or hydrolytically cleaved, resulting in release
of a Drug. The released Drug is then free to migrate in the cytosol
and induce cytotoxic activities. In an alternative embodiment, the
Drug is cleaved from the Compound of the Invention outside the
target cell, and the Drug subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to the infectious disease
cell.
In one embodiment, the infectious disease type of infectious
disease that the animal needs treatment or prevention of.
In one embodiment, the Compounds of the Invention kill or inhibit
the multiplication of cells that produce a particular infectious
disease.
Particular types of infectious diseases that can be treated with
the Compounds of the Invention include, but are not limited to,
those disclosed in Table 7.
TABLE-US-00010 TABLE 7 Bacterial Diseases: Diptheria Pertussis
Occult Bacteremia Urinary Tract Infection Gastroenteritis
Cellulitis Epiglottitis Tracheitis Adenoid Hypertrophy
Retropharyngeal Abcess Impetigo Ecthyma Pneumonia Endocarditis
Septic Arthritis Pneumococcal Peritonitis Bactermia Meningitis
Acute Purulent Meningitis Urethritis Cervicitis Proctitis
Pharyngitis Salpingitis Epididymitis Gonorrhea Syphilis Listeriosis
Anthrax Nocardiosis Salmonella Typhoid Fever Dysentery
Conjuntivitis Sinusitis Brucellosis Tullaremia Cholera Bubonic
Plague Tetanus Necrotizing Enteritis Actinomycosis Mixed Anaerobic
Infections Syphilis Relapsing Fever Leptospirosis Lyme Disease Rat
Bite Fever Tuberculosis Lymphadenitis Leprosy Chlamydia Chlamydial
Pneumonia Trachoma Inclusion Conjunctivitis Systemic Fungal
Diseases: Histoplamosis Coccicidiodomycosis Blastomycosis
Sporotrichosis Cryptococcsis Systemic Candidiasis Aspergillosis
Mucormycosis Mycetoma Chromomycosis Rickettsial Diseases: Typhus
Rocky Mountain Spotted Fever Ehrlichiosis Eastern Tick-Borne
Rickettsioses Rickettsialpox Q Fever Bartonellosis Parasitic
Diseases: Malaria Babesiosis African Sleeping Sickness Chagas'
Disease Leishmaniasis Dum-Dum Fever Toxoplasmosis
Meningoencephalitis Keratitis Entamebiasis Giardiasis
Cryptosporidiasis Isosporiasis Cyclosporiasis Microsporidiosis
Ascariasis Whipworm Infection Hookworm Infection Threadworm
Infection Ocular Larva Migrans Trichinosis Guinea Worm Disease
Lymphatic Filariasis Loiasis River Blindness Canine Heartworm
Infection Schistosomiasis Swimmer's Itch Oriental Lung Fluke
Oriental Liver Fluke Fascioliasis Fasciolopsiasis Opisthorchiasis
Tapeworm Infections Hydatid Disease Alveolar Hydatid Disease Viral
Diseases: Measles Subacute sclerosing panencephalitis Common Cold
Mumps Rubella Roseola Fifth Disease Chickenpox Respiratory
syncytial virus infection Croup Bronchiolitis Infectious
Mononucleosis Poliomyelitis Herpangina Hand-Foot-and-Mouth Disease
Bornholm Disease Genital Herpes Genital Warts Aseptic Meningitis
Myocarditis Pericarditis Gastroenteritis Acquired Immunodeficiency
Syndrome (AIDS) Reye's Syndrome Kawasaki Syndrome Influenza
Bronchitis Viral "Walking" Pneumonia Acute Febrile Respiratory
Disease Acute pharyngoconjunctival fever Epidemic
keratoconjunctivitis Herpes Simplex Virus 1 (HSV-1) Herpes Simples
Virus 2 (HSV-2) Shingles Cytomegalic Inclusion Disease Rabies
Progressive Multifocal Leukoencephalopathy Kuru Fatal Familial
Insomnia Creutzfeldt-Jakob Disease Gerstmann-Straussler-Scheinker
Disease Tropical Spastic Paraparesis Western Equine Encephalitis
California Encephalitis St. Louis Encephalitis Yellow Fever Dengue
Lymphocytic choriomeningitis Lassa Fever Hemorrhagic Fever
Hantvirus Pulmonary Syndrome Marburg Virus Infections Ebola Virus
Infections Smallpox
Multi-Drug Therapy of Infectious Diseases
The present invention also provides methods for treating an
infectious disease, comprising administering to an animal in need
thereof a Compound of the Invention and another therapeutic agent
that is an anti-infectious disease agent. In one embodiment, the
anti-infectious disease agent is, but not limited to, agents listed
in Table 8.
TABLE-US-00011 TABLE 8 Antibacterial Agents: .beta.-Lactam
Antibiotics: Penicillin G Penicillin V Cloxacilliin Dicloxacillin
Methicillin Nafcillin Oxacillin Ampicillin Amoxicillin
Bacampicillin Azlocillin Carbenicillin Mezlocillin Piperacillin
Ticarcillin Aminoglycosides: Amikacin Gentamicin Kanamycin Neomycin
Netilmicin Streptomycin Tobramycin Macrolides: Azithromycin
Clarithromycin Erythromycin Lincomycin Clindamycin Tetracyclines:
Demeclocycline Doxycycline Minocycline Oxytetracycline Tetracycline
Quinolones: Cinoxacin Nalidixic Acid Fluoroquinolones:
Ciprofloxacin Enoxacin Grepafloxacin Levofloxacin Lomefloxacin
Norfloxacin Ofloxacin Sparfloxacin Trovafloxicin Polypeptides:
Bacitracin Colistin Polymyxin B Sulfonamides: Sulfisoxazole
Sulfamethoxazole Sulfadiazine Sulfamethizole Sulfacetamide
Miscellaneous Antibacterial Agents: Trimethoprim Sulfamethazole
Chloramphenicol Vancomycin Metronidazole Quinupristin Dalfopristin
Rifampin Spectinomycin Nitrofurantoin Antiviral Agents: General
Antiviral Agents: Idoxuradine Vidarabine Trifluridine Acyclovir
Famicyclovir Pencicyclovir Valacyclovir Gancicyclovir Foscarnet
Ribavirin Amantadine Rimantadine Cidofovir Antisense
Oligonucleotides Immunoglobulins Inteferons Drugs for HIV
infection: Zidovudine Didanosine Zalcitabine Stavudine Lamivudine
Nevirapine Delavirdine Saquinavir Ritonavir Indinavir
Nelfinavir
Other Therapeutic Agents
The present methods can further comprise the administration of a
Compound of the Invention and an additional therapeutic agent or
pharmaceutically acceptable salts or solvates thereof. The Compound
of the Invention and the other therapeutic agent can act additively
or, more preferably, synergistically. In a preferred embodiment, a
composition comprising a Compound of the Invention is administered
concurrently with the administration of one or more additional
therapeutic agent(s), which can be part of the same composition or
in a different composition from that comprising the Compound of the
Invention. In another embodiment, a Compound of the Invention is
administered prior to or subsequent to administration of another
therapeutic agent(s).
In the present methods for treating cancer, an autoimmune disease
or an infectious disease, the other therapeutic agent can be an
antiemetic agent. Suitable antiemetic agents include, but are not
limited to, metoclopromide, domperidone, prochlorperazine,
promethazine, chlorpromazine, trimethobenzamide, ondansetron,
granisetron, hydroxyzine, acethylleucine monoethanolamine,
alizapride, azasetron, benzquinamide, bietanautine, bromopride,
buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol,
dolasetron, meclizine, methallatal, metopimazine, nabilone,
oxyperndyl, pipamazine, scopolamine, sulpiride,
tetrahydrocannabinols, thiethylperazine, thioproperazine and
tropisetron.
In another embodiment, the other therapeutic agent can be an
hematopoietic colony stimulating factor. Suitable hematopoietic
colony stimulating factors include, but are not limited to,
filgrastim, sargramostim, molgramostim and erythropoietin alfa.
In still another embodiment, the other therapeutic agent can be an
opioid or non-opioid analgesic agent. Suitable opioid analgesic
agents include, but are not limited to, morphine, heroin,
hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon,
apomorphine, normorphine, etorphine, buprenorphine, meperidine,
lopermide, anileridine, ethoheptazine, piminidine, betaprodine,
diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil,
levorphanol, dextromethorphan, phenazocine, pentazocine,
cyclazocine, methadone, isomethadone and propoxyphene. Suitable
non-opioid analgesic agents include, but are not limited to,
aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac,
fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin,
ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen,
piroxicam and sulindac.
The following examples are provided by way of illustration and not
limitation.
EXAMPLES
Materials and Methods. Commercially available reagents and solvents
were obtained as follows: HPLC-grade solvents, Fisher Scientific
(Atlanta, Ga.); anhydrous solvents, Aldrich (St. Louis, Mo.);
diisopropylazodicarboxylate (DIAD, 95%), Lancaster (Lancashire,
England); 4-aminobenzyl alcohol, Alfa Aesar (Ward Hill, Mass.);
L-citrulline, Novabiochem (Laufelfingen, Switzerland); all other
amino acids, Advanced ChemTech (Louisville, Ky.) or Novabiochem
(Laufelfingen, Switzerland); (1S,2R)-(+)-norephedrine and other
commercially available reagents, Aldrich or Acros; all coupling
reagents were acquired from Novabiochem or Aldrich. All solvents
used as reaction media are assumed to be anhydrous unless otherwise
indicated. 1H-NMR spectra were recorded on either a Varian Gemini
at 300 MHz or Varian Mercury 400 MHz spectrophotometer. Flash
column chromatography was performed using 230-400 mesh ASTM silica
gel from Fisher. Analtech silica gel GHLF plates were used for
thin-layer chromatography. Analytical HPLC was performed using a
Waters Alliance system using a photodiode array detector.
Preparative HPLC purification was performed using a Varian Prostar
system that had either a photodiode array or dual wavelength
detector. Combustion analyses were determined by Quantitative
Technologies, Inc., Whitehouse, N.J.
Examples 5-12 relate to Drugs that can be used as Drug units in the
invention.
Example 1
Preparation of Compound 21
##STR00174##
Fmoc-(L)-val-(L)-cit-PAB-OH (19) (14.61 g, 24.3 mmol, 1.0 eq., U.S.
Pat. No. 6,214,345 to Firestone et al.) was diluted with DMF (120
mL, 0.2 M) and to this solution was added a diethylamine (60 mL).
The reaction was monitored by HPLC and found to be complete in 2 h.
The reaction mixture was concentrated and the resulting residue was
precipitated using ethyl acetate (about 100 mL) under sonication
over for 10 min. Ether (200 mL) was added and the precipitate was
further sonicated for 5 min. The solution was allowed to stand for
30 min. without stirring and was then filtered and dried under high
vacuum to provide Val-cit-PAB-OH, which was used in the next step
without further purification. Yield: 8.84 g (96%). Val-cit-PAB-OH
(8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting
solution was treated with MC--OSu (Willner et al., Bioconjugate
Chem. 4, 521, 1993, 6.5 g, 21 mmol, 1.0 eq.). Reaction was complete
according to HPLC after 2 h. The reaction mixture was concentrated
and the resulting oil was precipitated using ethyl acetate (50 mL).
After sonicating for 15 min, ether (400 mL) was added and the
mixture was sonicated further until all large particles were broken
up. The solution was then filtered and the solid dried to provide
Compound 20 as an off-white solid. Yield: 11.63 g (96%); ES-MS m/z
757.9 [M-H]-
Compound 20 (8.0 g, 14.0 mmol) was diluted with DMF (120 mL, 0.12
M) and to the resulting solution was added
bis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and
diisopropylethylamine (3.66 mL, 21.0 mmol, 1.5 eq.). The reaction
was complete in 1 h according to HPLC. The reaction mixture was
concentrated to provide an oil that was precipitated with EtOAc,
and then triturated using EtOAc (about 25 mL). The solute was
further precipitated with ether (about 200 mL) and triturated for
15 min. The solid was filtered and dried under high vacuum to
provide Compound 21 which was 93% pure according to HPLC and used
in the next step without further purification. Yield: 9.7 g
(94%).
Example 2
Preparation of Compound 27
##STR00175##
Compound 26 (2.0 g, 2.31 mmol, 1.0 eq.) was diluted with
dichloromethane (30 mL), and to the resulting solution was added
bis(4-nitrophenyl)carbonate (2.72 g, 8.94 mmol, 3.8 eq.) followed
by diisopropylethylamine (1.04 mL, 5.97 mmol, 2.6 eq.). The
reaction was complete in 3 d, according to HPLC. The reaction
mixture was concentrated and the resulting residue was triturated
using ether, then filtered and dried under high vacuum to provide
Compound 27 as a yellow solid (2.37 g, 97%).
Example 3
Preparation of Compound 28
##STR00176##
Fmoc-phe-lys(Mtr)-OH (24) (0.5 g, 0.63 mmol, U.S. Pat. No.
6,214,345 to Firestone et al.) was diluted with dichloromethane to
a concentration of 0.5 M and to this solution was added
diethylamine in an amount that was approximately one-third of the
volume of the Compound 24/dichloromethane solution. The reaction
was allowed to stir and was monitored using HPLC. It was shown to
be complete by HPLC in 3 h. The reaction mixture was concentrated
in vacuo, and the resulting residue was diluted with ethyl acetate
and then reconcentrated. The resulting residue was triturated using
ether and filtered. The residual solid was diluted with
dichloromethane to a concentration of 0.2M, and to the resulting
solution was added MC--OSu (0.20 g, 0.63 mmol, 1.0 eq.) and
diisopropylethylamine (0.12 mL, 0.70 mmol, 1.1 eq.). The reaction
mixture was allowed to stir under a nitrogen atmosphere for 16 h,
after which time HPLC showed very little starting material. The
reaction mixture was then concentrated and the resulting residue
was triturated using ether to provide Compound 28 as a colored
solid. Yield: 100 mg (21%); ES-MS m/z 757.9 [M-H].sup.-.
Example 4
Preparation of Compound 19A
##STR00177##
Compound 19 (1.0 g, 1.66 mmol) was diluted with DMF (10 mL) and to
the resulting solution was added bis(4-nitrophenyl)carbonate (1.0
g, 3.3 mmol, 2.0 eq.).
The reaction mixture was immediately treated with
diisopropylethylamine (0.43 mL, 2.5 mmol, 1.5 eq.) and the reaction
was allowed to stir under an argon atmosphere. The reaction was
complete in 2.5 h according to HPLC. The reaction mixture was
concentrated to provide a light brown oil that was precipitated
using ethyl acetate (5 mL), then precipitated again using ether
(about 100 mL). The resulting precipitate was allowed to stand for
30 min, and was then filtered and dried under high vacuum to
provide Compound 19a as an off-white powder. Yield: 1.05 g (83%);
ES-MS m/z 767.2 [M+H].sup.+; UV .lamda..sub.max 215, 256 nm.
Example 5
Preparation of Compound 49
##STR00178##
Compound 49 was made according to General Procedure D using
Fmoc-Me-val-val-dil-O-t-Bu 39 (0.40 g, 0.57 mmol) as the tripeptide
and Boc-dap-nor 44 (0.26 g, 0.62 mmol, 1.1 eq.) as the dipeptide.
The reaction mixture was purified using flash column chromatography
(silica gel column, eluant--100% EtOAc). Two Fmoc-containing
products eluted: the Fmoc derivative of Compound 49 (R.sub.f 0.17
in 100% EtOAc) and what was believed to be the Fmoc derivative of
the TFA acetate of Compound 49 (R.sub.f 0.37). The products were
combined to provide a white foam that was subjected to General
Procedure E. Reaction was complete after 2 h. Solvents were removed
to provide an oil that was purified using flash column
chromatography (eluant--9:1 Dichloromethane-methanol) to provide
Compound 49.
Example 6
Preparation of Compound 50
##STR00179##
Compound 50 was prepared by reacting tripeptide 42 and dipeptide 48
according to General Procedure D using triethylamine (5.0 eq.) as
the base. After concentration of the reaction mixture, the
resulting residue was directly injected onto a reverse phase
preparative-HPLC column (Varian Dynamax column 21.4 mm.times.25 cm,
5.mu., 100 .ANG., using a gradient run of MeCN and 0.1M
TEA/CO.sub.2 at 20 mL/min from 10% to 100% over 40 min followed by
100% MeCN for 20 min). The relevant fractions were pooled and
concentrated, and the resulting residue was diluted with 10 mL of
dichloromethane-ether (1:1). The solution was cooled to 0.degree.
C. and 1.0M ethereal HCl was added dropwise (approx. 10 eq.). The
precipitate, Compound 50, was filtered and dried and was
substantially pure by HPLC. Yield: 71 mg (43%); ES-MS m/z 731.6
[M+H].sup.+; UV .lamda..sub.max 215, 238, 290 nm. Anal. Calc.
C.sub.40H.sub.70N.sub.6O.sub.6.4H.sub.2O.2HCl: C, 54.84; H, 9.20;
N, 9.59. Found: C, 55.12; H, 9.41; N, 9.82.
Example 7
Preparation of Compound 51
##STR00180##
Compound 51 was prepared by reacting Fmoc-tripeptide 41 and
dipeptide 46 according to General Procedure D using triethylamine
as the base. After concentration of the reaction mixture, the
residue was directly injected onto a reverse phase preparative-HPLC
column (Varian Dynamax column 21.4 mm.times.25 cm, 5.mu., 100
.ANG., using a gradient run of MeCN and 0.1M TEA/CO.sub.2 at 20
mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20
min). The relevant fractions were pooled and concentrated to
provide a white solid intermediate that was used in the next step
without further purification. ES-MS m/z 882.9 [M+NH.sub.4].sup.+,
899.9 [M+Na].sup.+; UV .lamda..sub.max 215, 256 nm.
Deprotection of the white solid intermediate was performed
according to General Procedure E. The crude product was purified
using preparative-HPLC (Varian Dynamax column 21.4 mm.times.25 cm,
5.mu., 100 .ANG., using a gradient run of MeCN and 0.1M
TEA/CO.sub.2 at 20 mL/min from 10% to 100% over 40 min followed by
100% MeCN for 20 min). The relevant fractions were pooled and
concentrated to provide Compound 51 as a sticky solid. ES-MS m/z
660.1 [M+H].sup.+, 682.5 [M+Na].sup.+; UV .lamda..sub.max 215
nm.
Example 8
Preparation of Compound 52
##STR00181##
Boc-dolaproine (0.33 g, 1.14 mmol) and
(1S,2S)-(-)-1,2-diphenylethylenediamine (0.5 g, 2.28 mmol, 2.0 eq.)
were diluted with dichloromethane, (10 mL) and to the resulting
solution was added triethylamine (0.32 mL, 2.28 mmol, 2.0 eq.),
then DEPC (0.39 mL, 2.28 mmol, 2.0 eq.). After 4 h, additional DEPC
(0.39 mL) was added and the reaction was allowed to stir overnight.
The reaction mixture was concentrated and the resulting residue was
purified using preparative-HPLC (Varian Dynamax C.sub.18 column
21.4 mm.times.25 cm, 5.mu., 100 .ANG., using a gradient run of MeCN
and water at 20 mL/min from 10% to 100% over 40 min followed by
100% MeCN for 20 min). The relevant fractions were pooled and
concentrated to provide a yellow gummy solid peptide intermediate
that was used without further purification. R.sub.f 0.15 (100%
EtOAc); ES-MS m/z 482.4 [M+H].sup.+; UV .lamda..sub.max 215, 256
nm.
The yellow gummy peptide intermediate (0.24 g, 0.50 mmol) was
diluted with dichloromethane, and to the resulting solution was
added diisopropylethylamine (0.18 mL, 1.0 mmol, 2.0 eq.) and
Fmoc-Cl (0.15 g, 0.55 mmol, 1.1 eq.). The reaction was allowed to
stir for 3 h, after which time HPLC showed a complete reaction. The
reaction mixture was concentrated to an oil, and the oil was
diluted with EtOAc and extracted successively with 10% aqueous
citric acid, water, saturated aqueous sodium bicarbonate, and
brine. The EtOAc layer was dried, filtered, and concentrated, and
the resulting residue was purified using flash column
chromatography (silica gel 230-400 mesh; eluant gradient 4:1
hexanes-EtOAc to 1:1 hexanes-EtOAc) to provide Compound 45 as a
white solid. Yield: 0.37 g (46% overall); R.sub.f 0.47 (1:1
hexanes-EtOAc); ES-MS m/z 704.5 [M+H].sup.+, 721.4
[M+NH.sub.4].sup.+; UV .lamda..sub.max 215, 256 nm.
Compound 52 was prepared by reacting tripeptide 42 (94 mg, 0.13
mmol) and dipeptide compound 45 (65 mg, 0.13 mmol) according to
General Procedure D (using 3.6 eq. of diisopropylethylamine as the
base). After concentration of the reaction mixture, the resulting
residue was diluted with EtOAc and washed successively with 10%
aqueous citric acid, water, saturated aqueous sodium bicarbonate,
and brine. The organic phase was dried, filtered and concentrated
to provide a white solid residue which was diluted with
dichloromethane and deprotected according to General Procedure E.
According to HPLC, reaction was complete after 2 h. The reaction
mixture was concentrated to an oil. The oil was diluted with DMSO,
and the resulting solution was purified using a reverse phase
preparative-HPLC (Varian Dynamax column 21.4 mm.times.25 cm, 5.mu.,
100 .ANG., using a gradient run of MeCN and 0.1% TFA at 20 mL/min
from 10% to 100% over 40 min followed by 100% MeCN for 20 min). Two
products having similar UV spectra were isolated. The major
product, Compound 52, was provided as an off-white solid. Overall
yield: 24 mg (23%); ES-MS m/z 793.5 [M+H].sup.+; UV .lamda..sub.max
215 nm.
Example 9
Preparation of Compound 53
##STR00182##
Boc-phenylalanine (1.0 g, 3.8 mmol) was added to a suspension of
1,4-diaminobenzene.HCl (3.5 g, 19.0 mmol, 5.0 eq.) in triethylamine
(10.7 mL, 76.0 mmol, 20 eq.) and dichloromethane (50 mL). To the
resulting solution was added DEPC (3.2 mL, 19.0 mmol, 5.0 eq.) via
syringe. HPLC showed no remaining Boc-phe after 24 h. The reaction
mixture was filtered, and the filtrate was concentrated to provide
a dark solid. The dark solid residue was partitioned between 1:1
EtOAc-water, and the EtOAc layer was washed sequentially with water
and brine. The EtOAc layer was dried and concentrated to provide a
dark brown/red residue that was purified using HPLC (Varian Dynamax
column 41.4 mm.times.25 cm, 5.mu., 100 .ANG., using a gradient run
of MeCN and water at 45 mL/min form 10% to 100% over 40 min
followed by 100% MeCN for 20 min). The relevant fractions were
combined and concentrated to provide a red-tan solid intermediate.
Yield: 1.4 g (100%); ES-MS m/z 355.9 [M+H].sup.+; UV
.lamda..sub.max 215, 265 nm; .sup.1H NMR (CDCl.sub.3) .delta. 7.48
(1H, br s), 7.22-7.37 (5H, m), 7.12 (2H, d, J=8.7 Hz), 7.61 (2H, d,
J=8.7 Hz), 5.19 (1H, br s), 4.39-4.48 (1H, m), 3.49 (2H, s), 3.13
(2H, d, J=5.7 Hz), 1.43 (9H, s).
The red-tan solid intermediate (0.5 g, 1.41 mmol) and
diisopropylethylamine (0.37 mL, 2.11 mmol, 1.5 eq.) were diluted
with dichloromethane (10 mL), and to the resulting solution was
added Fmoc-Cl (0.38 g, 1.41 mmol). The reaction was allowed to
stir, and a white solid precipitate formed after a few minutes.
Reaction was complete according to HPLC after 1 h. The reaction
mixture was filtered, and the filtrate was concentrated to provide
an oil. The oil was precipitated with EtOAc, resulting in a
reddish-white intermediate product, which was collected by
filtration and dried under vacuum. Yield: 0.75 g (93%); ES-MS m/z
578.1 [M+H].sup.+, 595.6 [M+NH.sub.4].sup.+.
The reddish-white intermediate (0.49 g, 0.85 mmol), was diluted
with 10 mL of dichloromethane, and then treated with 5 mL of
trifluoroacetic acid. Reaction was complete in 30 min according to
reverse-phase HPLC. The reaction mixture was concentrated and the
resulting residue was precipitated with ether to provide an
off-white solid. The off-white solid was filtered and dried to
provide an amorphous powder, which was added to a solution of
Boc-dap (0.24 g, 0.85 mmol) in dichloromethane (10 mL). To this
solution was added triethylamine (0.36 mL, 2.5 mmol, 3.0 eq.) and
PyBrop (0.59 g, 1.3 mmol, 1.5 eq.). The reaction mixture was
monitored using reverse-phase HPLC. Upon completion, the reaction
mixture was concentrated, and the resulting residue was diluted
with EtOAc, and sequentially washed with 10% aqueous citric acid,
water, saturated aqueous sodium bicarbonate, water, and brine. The
EtOAc layer was dried (MgSO.sub.4), filtered, and concentrated. The
resulting residue was purified using flash column chromatography
(silica gel) to provide Compound 47 as an off-white powder. Yield:
0.57 g (88%); ES-MS m/z 764.7 [M+NH.sub.4].sup.+; UV
.lamda..sub.max 215, 265 nm; .sup.1H NMR (DMSO-d.sub.6) .delta.
10.0-10.15 (1H, m), 9.63 (1H, br s), 8.42 (1/2H, d, J=8.4 Hz), 8.22
(1/2H, d, J=8.4 Hz), 7.89 (2H, d, J=7.2 Hz), 7.73 (2H, d, J=7.6
Hz), 7.11-7.55 (13H, m), 4.69-4.75 (1H, m) 4.46 (2H, d, J=6.8 Hz),
4.29 (1H, t, J=6.4 Hz), 3.29 (3H, s), 2.77-3.47 (7H, m), 2.48-2.50
(3H, m), 2.25 (2/3H, dd, J=9.6, 7.2 Hz), 1.41-1.96 (4H, m), 1.36
(9H, s), 1.07 (1H, d, J=6.4 Hz, rotational isomer), 1.00 (1H, d,
J=6.4 Hz, rotational isomer).
Tripeptide compound 42 (55 mg, 0.11 mmol) and dipeptide compound 47
(85 mg, 0.11 mmol) were reacted according to General Procedure D
(using 3.0 eq. of diisopropylethylamine). After concentration of
the reaction mixture, the resulting residue was diluted with EtOAc,
and washed sequentially with 10% aqueous citric acid, water,
saturated aqueous sodium bicarbonate, and brine. The EtOAc layer
was dried, filtered and concentrated to provide a yellow oil. The
yellow oil was diluted with dichloromethane (10 mL) and deprotected
according to General Procedure E. According to HPLC, reaction was
complete after 2 h. The reaction mixture was concentrated to
provide an oil. The oil was diluted with DMSO, and the DMSO
solution was purified using reverse phase preparative-HPLC (Varian
Dynamax column 21.4 mm.times.25 cm, 5.mu., 100 .ANG., using a
gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100%
over 40 min followed by 100% MeCN for 20 min). The relevant
fractions were combined and concentrated to provide Compound 53 as
an off-white solid. Overall yield: 42 mg (44% overall); ES-MS m/z
837.8 [M+H].sup.+, 858.5 [M+Na].sup.+; UV .lamda..sub.max 215, 248
nm.
Example 10
Preparation of Compound 54
##STR00183##
Compound 54 was prepared according to K. Miyazaki, et al. Chem.
Pharm. Bull. 1995, 43(10), 1706-18.
Example 11
Preparation of Compound 55
##STR00184##
Compound 55 was synthesized in the same manner as Compound 54, but
by substituting FmocMeVal-Ile-Dil-tBu (40) for
FmocMeVal-Val-Dil-tBu (39) as the starting material.
Example 12
Preparation of Compound 56
##STR00185##
Carbamic acid
[(1S)-1-(azidomethyl)-2-phenylethyl]-1,1-dimethylethyl ester (0.56
g, 2 mmol, prepared as described in J. Chem. Research (S), 1992,
391), was diluted with a 4 M solution of HCl in dioxane (10 mL) and
the resulting solution allowed to stir for 2 hr at room
temperature. Toluene (10 mL) was then added to the reaction, the
reaction mixture was concentrated and the resulting residue was
azeotropically dried under vacuum using toluene (3.times.15 mL), to
provide a white solid intermediate. ES-MS m/z 177.1
[M+H].sup.+.
The white solid intermediate was diluted with dichloromethane (5
mL) and to the resulting solution was added sequentially
N-Boc-Dolaproine (0.58 g, 1 eq.), triethylamine (780 .mu.L, 3 eq.)
and DEPC (406 .mu.L, 1.2 eq.), and the reaction mixture was allowed
to stir for 2 h at room temperature. Reaction progress was
monitored using reverse-phase HPLC. Upon completion of reaction as
determined by HPLC, the reaction mixture was diluted with
dichloromethane (30 mL), the dichloromethane layer was washed
successively with 10% aqueous citric acid (20 mL), saturated
aqueous NaHCO.sub.3 (20 mL), and water (20 mL). The dichloromethane
layer was concentrated and the resulting residue was purified via
flash column chromatography using a step gradient of 0-5% methanol
in dichloromethane. The relevant fractions were combined and
concentrated to provide a solid intermediate, 0.78 g (88%). ES-MS
m/z 446.1 [M+H].sup.+, 468.3 [M+Na].sup.+.
The solid intermediate (450 mg, 1 mmol) and Tripeptide 42 (534 mg,
1.1 eq.) were diluted with a 50% solution of TFA in dichloromethane
(10 mL), and the resulting reaction was allowed to stir for 2 h at
room temperature. Toluene (10 mL) was added to the reaction and the
reaction mixture was concentrated. The resulting amine intermediate
was azeotropically dried using toluene (3.times.20 mL) and dried
under vacuum overnight.
The resulting amine intermediate was diluted with dichloromethane
(2 mL) and to the resulting solution was added triethylamine (557
.mu.L, 4 eq.), followed by DEPC (203 .mu.L, 1.4 eq.). The reaction
mixture was allowed to stir for 4 h at room temperature and
reaction progress was monitored using HPLC. Upon completion of
reaction, the reaction mixture was diluted with dichloromethane (30
mL) and the dichloromethane layer was washed sequentially using
saturated aqueous NaHCO.sub.3 (20 mL) and saturated aqueous NaCl
(20 mL). The dichloromethane layer was concentrated and the
resulting residue was purified using flash column chromatography in
a step gradient of 0-5% methanol in dichloromethane. The relevant
fractions were combined and concentrated and the resulting residue
was dried using a dichloromethane:hexane (1:1) to provide a white
solid intermediate, 0.64 g (84%). ES-MS m/z 757.5 [M+H].sup.+.
The white solid intermediate (536 mg, 0.73 mmol) was diluted with
methanol and to the resulting solution was added 10% Pd/C (100 mg).
The reaction was placed under a hydrogen atmosphere and was allowed
to stir at atmospheric pressure and room temperature for 2 h.
Reaction progress was monitored by HPLC and was complete in 2 h.
The reaction flask was purged with argon and the reaction mixture
was filtered through a pad of Celite. The Celite pad was
subsequently washed with methanol (30 mL) and the combined
filtrates were were concentrated to yield a gray solid intermediate
which was used without further purification. Yield=490 mg (91%).
ES-MS m/z 731.6 [M+H].sup.+, 366.6 [M+2H].sup.2+/2.
The gray solid intermediate (100 mg, 0.136 mmol),
N-Boc-4-aminobenzoic acid (39 mg, 1.2 eq.) and triethylamine (90
.mu.L, 4 eq.) were diluted with dichloromethane (2 mL) and to the
resulting solution was added DEPC (28 .mu.L, 1.2 eq.). The reaction
mixture was allowed to stir at room temperature for 2 h, then the
reaction mixture was diluted with dichloromethane (30 mL). The
dichloromethane layer was sequentially washed with saturated
aqueous NaHCO.sub.3 (20 mL) and saturated aqueous NaCl (20 mL). The
dichloromethane layer was then concentrated and the resulting
residue was purified via flash column chromatography using a step
gradient of 0-5% in dichlormethane. The relevant fractions were
combined and concentrated and the resulting residue was dried using
dichloromethane:hexane (1:1) to provide a white solid intermediate.
ES-MS m/z 950.7 [M+H].sup.+.
The white solid intermediate was diluted with a 50% solution of TFA
in dichloromethane and allowed to stir for 2 h at room temperature.
Toluene (10 mL) was added to the reaction and the reaction mixture
was concentrated. The resulting residue was azeotropically dried
using toluene (3.times.15 mL), to provide a yellow oil which was
purified using preparative HPLC (C.sub.18-RP Varian Dynamax column,
5.mu., 100 .ANG., linear gradient of MeCN from 10 to 95% in 0.05 M
Triethylammonium carbonate buffer, pH 7.0, in 30 min at a flow rate
of 10 mL/min). The relevant fractions were combined and
concentrated and the resulting residue was azeotropically dried
using MeCN (3.times.20 mL), to provide Compound 56 as white solid:
101 mg (87% over 2 steps). ES-MS m/z 850.6 [M+H].sup.+, 872.6
[M+Na].sup.+.
Example 13
Preparation of Compound 57
##STR00186##
Compound 49 (100 mg, 0.14 mmol), Compound 27 (160 mg, 0.15 mmol,
1.1 eq.), and HOBt (19 mg, 0.14 mmol, 1.0 eq.) were diluted with
DMF (2 mL). After 2 min, pyridine (0.5 mL) was added and the
reaction mixture was monitored using reverse-phase HPLC. Neither
Compound 49 nor Compound 27 was detected after 24 h. The reaction
mixture was concentrated, and the resulting residue was purified
using reverse phase preparative-HPLC (Varian Dynamax column 21.4
mm.times.25 cm, 5.mu., 100 .ANG., using a gradient run of MeCN and
Et.sub.3N--CO.sub.2 (pH 7) at 20 mL/min from 10% to 100% over 40
min followed by 100% MeCN for 20 min). The relevant fractions were
pooled and concentrated to provide an off-white solid intermediate.
ES-MS m/z 1608.7 [M+H].sup.+
The off-white solid intermediate was diluted with MeCN/water/TFA in
an 85:5:10 ratio, respectively. The reaction mixture was monitored
using HPLC and was complete in 3 h. The reaction mixture was
directly concentrated and the resulting residue was purified using
reverse phase preparative-HPLC (Varian Dynamax column 21.4
mm.times.25 cm, 5.mu., 100 .ANG., using a gradient run of MeCN and
0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100%
MeCN for 20 min). The relevant fractions were combined and
concentrated to provide Compound 57 as an off-white powder. Yield:
46 mg (32% overall); ES-MS m/z 1334.8 [M+H].sup.+; UV
.lamda..sub.max 215, 256 nm.
Example 14
Preparation of Compound 58
##STR00187##
Compound 49 (1.69 g, 2.35 mmol), Compound 21 (2.6 g, 3.52 mmol, 1.5
eq.), and HOBt (64 mg, 0.45 mmol, 0.2 eq.) were diluted with DMF
(25 mL). After 2 min, pyridine (5 mL) was added and the reaction
was monitored using reverse-phase HPLC. The reaction was shown to
be complete in 24 h. The reaction mixture was concentrated to
provide a dark oil, which was diluted with 3 mL of DMF. The DMF
solution was purified using flash column chromatography (silica
gel, eluant gradient:100% dichloromethane to 4:1
dichloromethane-methanol). The relevant fractions were combined and
concentrated to provide an oil that solidified under high vacuum to
provide a mixture of Compound 58 and unreacted Compound 49 as a
dirty yellow solid (R.sub.f 0.40 in 9:1 dichloromethane-methanol).
The dirty yellow solid was diluted with DMF and purified using
reverse-phase preparative-HPLC (Varian Dynamax C.sub.18 column 41.4
mm.times.25 cm, 8 m, 100 .ANG., using a gradient run of MeCN and
0.1% aqueous TFA at 45 mL/min from 10% to 100% over 40 min followed
by 100% MeCN for 20 min) to provide Compound 58 as an amorphous
white powder (Rf 0.40 in 9:1 dichloromethane-methanol) which was
>95% pure by HPLC and which contained less than 1% of Compound
49. Yield: 1.78 g (57%); ES-MS m/z 1316.7 [M+H].sup.+; UV
.lamda..sub.max 215, 248 nm.
Example 15
Preparation of Compound 59
##STR00188##
The hydrochloride salt of Compound 51 (11 mg, 15.2 mmol) and
Compound 21 (11 mg, 15.2 mmol) were diluted with
1-methyl-2-pyrollidinone (1 mL) and to the resulting solution was
added diisopropylethylamine (5.3 mL, 30.3 mmol, 2.0 eq.). The
mixture was allowed to stir under argon atmosphere for 3 d while
being monitored using HPLC. After this time, much unreacted
starting material still remained, HOBt (1.0 eq.) was added and the
reaction mixture was allowed to stir for 24 h, after which time no
starting material remained according to HPLC. The reaction mixture
was concentrated and the resulting residue was purified using
preparative-HPLC (Varian Dynamax C.sub.18 column 21.4 mm.times.25
cm, 5 m, 100 .ANG., using a gradient run of MeCN and water at 20
mL/min from 10% to 100% over 30 min followed by 100% MeCN for 20
min). The relevant fractions were combined and concentrated to
provide Compound 59 as a white solid. Yield: 13 mg (67%); ES-MS m/z
1287.2 [M+H].sup.+, 1304.3 [M+NH.sub.4].sup.+; UV .lamda..sub.max
215, 248 nm
Example 16
Preparation of Compound 60
##STR00189##
Compound 53 (9 mg, 10.8 .mu.mol) and Compound 28 (5.2 mg, 10.8
.mu.mol) were diluted with dichloromethane (1 mL) and to the
resulting solution was added HATU (6.3 mg, 16.1 .mu.mol, 1.5 eq.),
followed by pyridine (1.3 .mu.L, 16.1 .mu.mol, 1.5 eq.). The
reaction mixture was allowed to stir under argon atmosphere while
being monitored using HPLC. The reaction was complete after 6 h.
The reaction mixture was concentrated and the resulting residue was
diluted with DMSO. The DMSO solution was purified using reverse
phase preparative-HPLC (Varian Dynamax column 21.4 mm.times.25 cm,
5.mu., 100 .ANG., using a gradient run of MeCN and 0.1% TFA at 20
mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20
min) and the relevant fractions were combined and concentrated to
provide an an off-white solid intermediate which was >95% pure
according to HPLC.
The off-white solid intermediate was diluted with dichloromethane
(2 mL) and the resulting solution was treated with TFA (0.5 mL).
The reaction was monitored using HPLC, and was complete in 2 h. The
reaction mixture was concentrated, and the resulting residue was
diluted with DMSO and purified under the same conditions as
described in Example 13. The relevant fractions were combined and
concentrated to provide Compound 60 as an off-white powder. Yield:
14.9 mg (90%); ES-MS m/z 1304.6 [M+H].sup.+; UV .lamda..sub.max
215, 275 nm.
Example 17
Preparation of Compound 61
##STR00190##
The trifluoroacetate salt of Compound 53 (0.37 g, 0.39 mmol, 1.0
eq.) and Compound 18 (0.30 g, 0.58 mmol, 1.5 eq.) were diluted with
DMF (5 mL, 0.1 M), and to the resulting solution was added pyridine
(95 .mu.L, 1.2 mmol, 3.0 eq.). HATU (0.23 g, 0.58 mmol, 1.5 eq.) wa
then added as a solid and the reaction mixture was allowed to stir
under argon atmosphere while being monitored using HPLC. The
reaction progressed slowly, and 4 h later, 1.0 eq. of
diisopropylethylamine was added. Reaction was complete in 1 h. The
reaction mixture was concentrated in vacuo and the resulting
residue was purified using preparative-HPLC (Varian Dynamax C18
column 41.4 mm.times.25 cm, 5.mu., 100 .ANG., using a gradient run
of MeCN and 0.1% aqueous TFA at 45 mL/min from 10% to 100% over 40
min followed by 100% MeCN for 20 min) to provide a faint pink solid
intermediate.
The pink solid intermediate was diluted with DMF (30 mL) and to the
resulting solution was added diethylamine (15 mL). Reaction was
complete by HPLC in 2 h. The reaction mixture was concentrated and
the resulting residue was washed twice with ether. The solid
intermediate was dried under high vacuum and then used directly in
the next step.
The solid intermediate was diluted with DMF (20 mL) and to the
resulting solution was added MC--OSu (0.12 g, 0.39 mmol, 1.0 eq.).
After 4 d, the reaction mixture was concentrated to provide an oil
which was purified using preparative-HPLC (Varian Dynamax C18
column 41.4 mm.times.25 cm, 5.mu., 100 .ANG., using a gradient run
of MeCN and 0.1% aqueous TFA at 45 mL/min from 10% to 100% over 40
min followed by 100% MeCN for 20 min). Compound 61 was isolated as
a white flaky solid. Yield: 0.21 g (38% overall); ES-MS m/z 1285.9
[M+H]+; 13.07.8 [M+Na]+; UV .lamda..sub.max 215, 266 nm.
Example 18
Preparation of Compound 62
##STR00191##
Fmoc-val-cit-PAB-OCO-Pnp (19a) (0.65 g, 0.85 mmol, 1.1 eq.),
Compound 49 (0.55 g, 0.77 mmol, 1.0 eq.), and HOBt (21 mg, 0.15
mmol, 2.0 eq.) were diluted with DMF (2.0 mL) and dissolved using
sonication. To the resulting solution was added pyridine (0.5 mL)
and the reaction was monitored using HPLC. After 24 h,
diisopropylethylamine (1.0 eq.) was added and the reaction was
allowed to stand without stirring for 24 h. The reaction mixture
was concentrated to provide an oil residue. The oil residue was
purified using reverse phase preparative-HPLC (Varian Dynamax
column 41.4 mm.times.25 cm, 5.mu., 100 .ANG., using a gradient run
of MeCN and 0.1% TFA at 45 mL/min from 10% to 100% over 40 min
followed by 100% MeCN for 20 min.) The desired fractions were
pooled and concentrated to yield an oil that was precipitated with
ether to provide an off-white solid intermediate. Yield: 0.77 g
(74%); ES-MS m/z 1345.7 [M+H].sup.+; UV .lamda..sub.max 215, 254
nm.
The off-white solid intermediate (about 85 mg) was deprotected
using diethylamine (1 mL) in DMF (3 mL). After 1 h, the reaction
was complete. The reaction mixture was concentrated, and the
resulting residue was precipitated in 1 mL of EtOAc followed by
addition of excess ether (about 20 mL). The amine intermediate was
filtered and dried under high vacuum and used in the next step
without further purification.
The amine intermediate (70 mg, 61 .mu.mol, 1.0 eq.) was taken up in
DMF (10 mL), and to the resulting solution was added sequentially,
bromoacetamidocaproic acid (17 mg, 67 .mu.mol, 1.1. eq.), PyBrop
(32 mg, 67 .mu.mol, 1.1 eq.), and diisopropylethylamine (16 .mu.L,
92 .mu.mol, 1.5 eq.). After 24 h, an additional 1.0 eq. of
bromoacetamidocaproic acid was added. Reaction was stopped after 30
h. The reaction mixture was concentrated to an oil and the oil
purified using reverse phase preparative-HPLC (Synergi MaxRP
C.sub.12 column 21.4 mm.times.25 cm, 5.mu., 80 .ANG., using a
gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100%
over 40 min followed by 100% MeCN for 20 min.). The relevant
fractions were combined and concentrated to provide Compound 62 as
a white solid. Yield: 23 mg (27%); ES-MS m/z 1356.7 [M+H].sup.+; UV
.lamda..sub.max 215, 247 nm.
Example 19
Preparation of Compound 63
##STR00192##
Fmoc-val-cit-PAB-OC(O)-Me-val-val-dil-dap-nor (about 48 mg,
obtained according to Example 18) was subjected to Fmoc-removal by
treating with diethylamine (1 mL) in DMF (3 mL). After 1 h, the
reaction was complete. The reaction mixture was concentrated and
the resulting residue was precipitated using 1 mL of EtOAc followed
by addition of excess ether (about 20 mL). The amine intermediate
was filtered and dried under high vacuum and used in the next step
without further purification.
The amine intermediate (35 .mu.mol, 1.1 eq.) was diluted with DMF
(2 mL), and to the resulting solution was added sequentially
maleimido-PEG acid (Frisch, B.; Boeckler, C.; Schuber, F.
Bioconjugate Chem. 1996, 7, 180-6; 7.8 mg, 32 .mu.mol, 1.0 eq.),
DEPC (10.7 .mu.L, 64 .mu.mol, 2.0 eq.), and diisopropylethylamine
(11.3 .mu.L, 64 .mu.mol, 2.0 eq.). The reaction was complete in 15
min according to HPLC. The reaction mixture was concentrated to
provide an oil. The oil was diluted with 1 mL of DMSO and purified
using reverse phase preparative-HPLC (Synergi MaxRP C.sub.12 column
21.4 mm.times.25 cm, 5.mu., 80 .ANG., using a gradient run of MeCN
and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by
100% MeCN for 20 min). The relevant fractions were combined and
concentrated to provide Compound 63 as a white solid.
Yield: 16.2 mg (34%); ES-MS m/z 1348.6 [M+H].sup.+; UV
.lamda..sub.max 215, 247 nm.
Examples 20-25 describe the conjugation of the monoclonal
antibodies cBR96 and cAC10 to a Drug-Linker Compound. These
antibodies were obtained as described in Bowen, et al., J. Immunol.
1993, 151, 5896; and Trail, et al., Science 1993, 261, 212,
respectively.
The number of Drug-Linker moities per Ligand in a
Drug-Linker-Ligand Conjugate varies from conjugation reaction to
conjugation reaction, but typically ranges from about 7 to about 9,
particularly when the Ligand is cBR96 or cAC10.
Example 20
Preparation of Compound 64
##STR00193##
cBR96 Antibody (24 mg) was reduced using DTT as described in
General Procedure L, then the number of thiols per antibody and the
antibody concentration were determined as described in General
Procedure M and General Procedure N, respectively. Result: [Ab]=4.7
mg/mL=29.4 .mu.M; [thiol]=265 .mu.M; SH/Ab=9.0 (Typical SH/Ab range
is from about 7.8 to about 9.5). Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added
to 4.2 mL of reduced antibody and the resulting solution was cooled
to 0.degree. C. using an ice bath. In a separate flask, a 130.5
.mu.L stock solution of Compound 57 (8.4 mM in DMSO, 8.5 mol
Compound 57 per mol reduced antibody) was diluted with MeCN (1.48
mL, pre-chilled to 0.degree. C. in an ice bath). The MeCN solution
of Compound 57 was rapidly added to the antibody solution and the
reaction mixture was stirred using a vortex instrument for 5-10
sec., returned to the ice bath and allowed to stir at 0.degree. C.
for 1 hr, after which time 218 .mu.L of a cysteine solution (100 mM
in PBS/DTPA) was then added to quench the reaction. 60 .mu.L of the
quenched reaction mixture was saved as a "qrm" sample.
While the reaction proceeded, three PD10 columns (Sephadex G25,
available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold
room and equilibrated with PBS (which had been pre-cooled to
0.degree. C. using an ice bath).
The quenched reaction mixture, which contained Compound 64, was
concentrated to .ltoreq.3 mL by ultracentrifugation using two
Ultrafree 4 centrifuge filtering devices (30K molecular weight
cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to
manufacturer's instructions) which were pre-cooled to 4.degree. C.
in a refrigerator and the concentrated reaction mixture was eluted
through the three pre-chilled PD10 columns using PBS as the eluent
(1 mL for each column). The eluted conjugate was collected in a
volume of 1.4 mL per column, for a total eluted volume of 4.2 mL.
The eluted Conjugate solution was then filtered using a sterile 0.2
micron syringe-end filter, 250 .mu.L of Conjugate solution was set
aside for analysis, and the remainder of the Conjugate solution was
frozen in sterile vials.
The concentration of Compound 64, the number of Drug molecules per
Antibody, the amount of quenched Drug-Linker and the percent of
aggregates were determined using General Procedures P, N, O and Q,
respectively. Assay Results: [Compound 64]=3.8 mg/mLg %
Aggregate=trace Residual Thiol Titration:Residual thiols=1.7/Ab.
Drug/Ab.about.9.0-1.7=7.3 Quenched Drug-Linker: undetectable Yield:
4.2 mL, 16 mg, 66%.
Example 21
Preparation of Compounds 65
##STR00194##
cAC10 Antibody (24 mg) was reduced using DTT as described in
General Procedure L, then the number of thiols per antibody and the
antibody concentration were determined as described in General
Procedure M and General Procedure N, respectively. Results:
[Ab]=4.9 mg/mL=30.7 .mu.M; [thiol]=283 .mu.M; 9.2 SH/Ab
Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added
to 4.2 mL of reduced antibody and the resulting solution was cooled
to 0.degree. C. using an ice bath. In a separate flask, 125 .mu.L
of a stock solution of Compound 57 (8.4 mM in DMSO, 8.5 mol
Compound 57 per mol reduced antibody) was diluted with MeCN (1.48
mL, pre-chilled to 0.degree. C. in an ice bath). The MeCN solution
of Compound 57 was rapidly added to the antibody solution and the
reaction mixture was stirred using a vortex instrument for 5-10
sec., then returned to the ice bath and allowed to stir at
0.degree. C. for 1 hr, after which time 218 .mu.L of a cysteine
solution (100 mM in PBS/DTPA) was then added to quench the
reaction. 60 .mu.L of the quenched reaction mixture was saved as a
"qrm" sample.
While the reaction proceeded, four PD10 columns (Sephadex G25,
available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold
room and equilibrated with PBS (which had been pre-cooled to
0.degree. C. using an ice bath).
The quenched reaction mixture, which contained Compound 65, was
concentrated to .ltoreq.3 mL by ultracentrifugation using two
Ultrafree 4 centrifuge filtering devices (30K molecular weight
cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to
manufacturer's instructions) which were pre-cooled to 4.degree. C.
in a refrigerator and the concentrated reaction mixture was eluted
through the four pre-chilled PD10 columns using PBS as the eluent
(1 mL for each column). The eluted conjugate was collected in a
volume of 1.4 mL per column, for a total eluted volume of 5.6 mL.
The eluted Conjugate solution was then filtered using a sterile 0.2
micron syringe-end filter, 250 .mu.L of Conjugate solution was set
aside for analysis, and the remainder of the Conjugate solution was
frozen in sterile vials.
The concentration of Compound 65, the number of Drug molecules per
Antibody, the amount of quenched Drug-Linker and the percent of
aggregates were then determined using General Procedures P, N, O
and Q, respectively. Assay Results: [Compound 65]=2.8 mg/mL %
Aggregate=trace Residual Thiol Titration:Residual thiols=1.6/Ab.
Drug/Ab.about.9.2-1.6=7.6 Not covalently bound Drug-Linker:
undetectable Yield: 5.6 mL, 15.7 mg, 65%.
Example 22
Preparation of Compound 66
##STR00195##
cBR96 Antibody (24 mg) was was reduced using DTT as described in
General Procedure L, then the number of thiols per antibody and the
antibody concentration were determined as described in General
Procedure M and General Procedure N, respectively. Result: [Ab]=3.7
mg/mL=23.1 .mu.M; [thiol]=218 .mu.M; 9.4 SH/Ab Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added
to 4.2 mL of reduced antibody and the resulting solution was cooled
to 0.degree. C. using an ice bath. In a separate flask, 145.5 .mu.L
of a stock solution of Compound 58 (8.3 mM in DMSO, 9.0 mol
Compound 58 per mol reduced antibody) was diluted with MeCN (1.48
mL, pre-chilled to 0.degree. C. in an ice bath). The MeCN solution
of Compound 58 was rapidly added to the antibody solution and the
reaction mixture was stirred using a vortex instrument for 5-10
sec., then returned to the ice bath and allowed to stir at
0.degree. C. for 1 hr, after which time 249 .mu.L of a cysteine
solution (100 mM in PBS/DTPA) was then added to quench the
reaction. 60 .mu.L of the quenched reaction mixture was saved as a
"qrm" sample.
While the reaction proceeded, three PD10 columns (Sephadex G25,
available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold
room and equilibrated with PBS (which had been pre-cooled to
0.degree. C. using an ice bath).
The quenched reaction mixture, which contained Compound 66, was
concentrated to .ltoreq.3 mL by ultracentrifugation using two
Ultrafree 4 centrifuge filtering devices (30K molecular weight
cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to
manufacturer's instructions) which were pre-cooled to 4.degree. C.
in a refrigerator and the concentrated reaction mixture was eluted
through the three pre-chilled PD10 columns using PBS as the eluent
(1 mL for each column). The eluted conjugate was collected in a
volume of 1.4 mL per column, for a total eluted volume of 4.2 mL.
The eluted Conjugate solution was then filtered using a sterile 0.2
micron syringe-end filter, 250 .mu.L of Conjugate solution was set
aside for analysis, and the remainder of the Conjugate solution was
frozen in sterile vials.
The concentration of Compound 66, the number of Drug molecules per
Antibody, the amount of quenched Drug-Linker and the percent of
aggregates were determined using General Procedures P, N, O and Q,
respectively. Assay Results: [Compound 66]=3.0 mg/mL %
Aggregate=trace Residual Thiol Titration:Residual thiols=0.4/Ab.
Drug/Ab.about.9.5-0.4=9.1 Not covalently bound Drug-Linker: 0.3% of
57-Cys adduct Yield: 5.3 mL, 15.9 mg, 66%.
Example 23
Preparation of Compound 67
##STR00196##
cAC10 Antibody (24 mg) was reduced using DTT as described in
General Procedure L, then the number of thiols per antibody and the
antibody concentration were determined as described in General
Procedure M and General Procedure N, respectively. Result: [Ab]=3.9
mg/mL=24.5 .mu.M; [thiol]=227 .mu.M; 9.3 SH/Ab Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added
to 4.2 mL of reduced antibody and the resulting solution was cooled
to 0.degree. C. using an ice bath. In a separate flask, 154.4 .mu.L
of a stock solution of Compound 58 (8.3 mM in DMSO, 9.0 mol
Compound 58 per mol reduced antibody) was diluted with MeCN (1.46
mL, pre-chilled to 0.degree. C. in an ice bath). The MeCN solution
of Compound 58 was rapidly added to the antibody solution and the
reaction mixture was stirred using a vortex instrument for 5-10
sec., then returned to the ice bath and allowed to stir at
0.degree. C. for 1 hr, after which time 249 .mu.L of a cysteine
solution (100 mM in PBS/DTPA) was then added to quench the
reaction. 60 .mu.L of the quenched reaction mixture was saved as a
"qrm" sample.
While the reaction proceeded, four PD10 columns (Sephadex G25,
available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold
room and equilibrated with PBS (which had been pre-cooled to
0.degree. C. using an ice bath).
The quenched reaction mixture, which contained Compound 67, was
concentrated to .ltoreq.3 mL by ultracentrifugation using two
Ultrafree 4 centrifuge filtering devices (30K molecular weight
cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to
manufacturer's instructions) which were pre-cooled to 4.degree. C.
in a refrigerator and the concentrated reaction mixture was eluted
through the four pre-chilled PD10 columns using PBS as the eluent
(1 mL for each column). The eluted conjugate was collected in a
volume of 1.4 mL per column, for a total eluted volume of 5.6 mL.
The eluted Conjugate solution was then filtered using a sterile 0.2
micron syringe-end filter, 250 .mu.L of Conjugate solution was set
aside for analysis, and the remainder of the Conjugate solution was
frozen in sterile vials.
The concentration of Compound 67, the number of Drug molecules per
Antibody, the amount of quenched Drug-Linker and the percent of
aggregates were determined using General Procedures P, N, O and Q,
respectively. Assay Results: [Compound 67]=3.0 mg/mL %
Aggregate=trace Residual Thiol Titration:Residual thiols=0.5/Ab.
Drug/Ab.about.9.5-0.5=9.0 Quenched Drug-Linker: 1.1% of 58-Cys
adduct Yield: 5.3 mL, 15.9 mg, 66%.
Example 24
Preparation of Compound 68
##STR00197##
cBR96 Antibody (24 mg) was reduced using DTT as described in
General Procedure L, then the number of thiols per antibody and the
antibody concentration were determined as described in General
Procedure M and General Procedure N, repectively. Result: [Ab]=4.4
mg/mL=27.2 .mu.M; [thiol]=277 .mu.M; 10.2 SH/Ab Conjugation:
The reduced antibody was diluted with DMSO (1.47 mL, pre-chilled to
0.degree. C. in an ice bath) so that the resulting solution was 20%
DMSO. The solution was allowed to stir for 10 min. at 0.degree. C.,
then 127.8 .mu.L of a stock solution of Compound 60 (7.6 mM
solution in DMSO; 9 mol Compound 60 per mol antibody) was rapidly
added. The reaction mixture was immediately stirred using a vortex
instrument and return to the ice bath and allowed to stir at
0.degree. C. for 1 hr, after which time 213 .mu.L, of a cysteine
solution (100 mM in PBS/DTPA) was then added to quench the
reaction. 60 .mu.L of the quenched reaction mixture was saved as a
"qrm" sample.
While the reaction proceeded, four PD10 columns (Sephadex G25,
available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold
room and equilibrated with PBS (which had been pre-cooled to
0.degree. C. using an ice bath).
The quenched reaction mixture, which contained Compound 68, was
concentrated to .ltoreq.3 mL by ultracentrifugation using two
Ultrafree 4 centrifuge filtering devices (30K molecular weight
cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to
manufacturer's instructions) which were pre-cooled to 4.degree. C.
in a refrigerator and the concentrated reaction mixture was eluted
through the four pre-chilled PD10 columns using PBS as the eluent
(1 mL for each column). The eluted conjugate was collected in a
volume of 1.4 mL per column, for a total eluted volume of 5.6 mL.
The eluted Conjugate solution was then filtered using a sterile 0.2
micron syringe-end filter, 250 .mu.L of Conjugate solution was set
aside for analysis, and the remainder of the Conjugate solution was
frozen in sterile vials.
The concentration of Compound 68, the number of Drug molecules per
Antibody, the amount of quenched Drug-Linker and the percent of
aggregates were determined using General Procedures P, N, O and Q,
respectively.
Because the absorbances of Compound 60 and antibody largely
overlap, spectrophotometric determination of the conjugate
concentration requires the measurement of absorbance at 270 and 280
nm. The molar concentration of conjugate is given by the following
formula:
[Conjugate]=(OD.sub.280.times.1.08e.sup.-5-OD.sub.270.times.8.20e.sup.-6)-
.times.dilution factor, where the values 1.08e.sup.-5 and
8.20e.sup.-6 are calculated from the molar extinction coefficients
of the drug and the antibody, which are estimated as:
.epsilon..sub.270 Compound 60=2.06e4 .epsilon..sub.270 cBR96=1.87e5
.epsilon..sub.280 Compound 60=1.57e4 .epsilon..sub.280 cBR96=2.24e5
Assay Results: [Compound 68]=3.2 mg/mL % Aggregate=trace Residual
Thiol Titration:Residual thiols=1.0/Ab. Drug/Ab.about.10.2-1.0=9.2
Quenched Drug-Linker: trace Yield: 5.6 mL, 17.9 mg, 75%.
Example 25
Preparation of Compound 69
##STR00198##
cAC10 Antibody (24 mg) was reduced using DTT as described in
General Procedure L, then the number of thiols per antibody and the
antibody concentration were determined as described in General
Procedure M and General Procedure N, repectively. Result: [Ab]=4.8
mg/mL=29.8 .mu.M; [thiol]=281 .mu.M; 9.4 SH/Ab Conjugation:
The reduced antibody was diluted with DMSO (1.47 mL, pre-chilled to
0.degree. C. in an ice bath) so that the resulting solution was 20%
DMSO. The solution was allowed to stir for 10 min. at 0.degree. C.,
then 140 .mu.L of a stock solution of Compound 60 (7.6 mM solution
in DMSO; 8.5 mol Compound 60 per mol antibody) was rapidly added.
The reaction mixture was immediately stirred using a vortex
instrument and return to the ice bath and allowed to stir for 1 hr
at 0.degree. C., after which time 213 .mu.L of a cysteine solution
(100 mM in PBS/DTPA) was then added to quench the reaction. 60
.mu.L of the quenched reaction mixture was saved as a "qrm"
sample.
While the reaction proceeded, four PD10 columns (Sephadex G25,
available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold
room and equilibrated with PBS (which had been pre-cooled to
0.degree. C. using an ice bath).
The quenched reaction mixture, which contained Compound 69, was
concentrated to .ltoreq.3 mL by ultracentrifugation using two
Ultrafree 4 centrifuge filtering devices (30K molecular weight
cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to
manufacturer's instructions) which were pre-cooled to 4.degree. C.
in a refrigerator and the concentrated reaction mixture was eluted
through the four pre-chilled PD10 columns using PBS as the eluent
(1 mL for each column). The eluted conjugate was collected in a
volume of 1.4 mL per column, for a total eluted volume of 5.6 mL.
The eluted Conjugate solution was then filtered using a sterile 0.2
micron syringe-end filter, 250 .mu.L of Conjugate solution was set
aside for analysis, and the remainder of the Conjugate solution was
frozen in sterile vials.
The concentration of Compound 69, the number of Drug molecules per
Antibody, the amount of quenched Drug-Linker and the percent of
aggregates were determined using General Procedures P, N, O and Q,
respectively.
Because the absorbances of Compound 60 and antibody largely
overlap, spectrophotometric determination of the conjugate
concentration requires the measurement of absorbance at 270 and 280
nm. The molar concentration of conjugate is given by the following
formula:
[Conjugate]=(OD.sub.280.times.1.08e.sup.-5-OD.sub.270.times.8.20e.sup.-6)-
.times.dilution factor, where the values 1.08e.sup.-5 and
8.20e.sup.-6 are calculated from the molar extinction coefficients
of the drug and the antibody, which are estimated as:
.epsilon..sub.270 Compound 60=2.06e.sup.4 .epsilon..sub.270
cAC10=2.10e.sup.5 .delta..sub.280 Compound 60=1.57e.sup.4
.epsilon..sub.280 cAC10=2.53e.sup.5 Assay Results: [Compound
69]=3.0 mg/mL % Aggregate=trace Residual Thiol Titration:Residual
thiols=0.7/Ab. Drug/Ab.about.9.4-0.7=8.7 Quenched Drug-Linker:
trace Yield: 5.6 mL, 16.8 mg, 70%.
Example 26
Preparation of Compound 75
##STR00199##
Diethyl (4-nitrobenzyl)phosphonate (1.1 g, 4.02 mmol) was diluted
in anhydrous THF (4 mL) and the resulting mixture was cooled to
0.degree. C. Sodium hydride (0.17 g, 4.22 mmol, 1.05 eq., 60%
dispersion in mineral oil) was added and the resulting reaction was
allowed to stir for 5 min. At this time gas evolution from the
reaction mixture had ceased. 2,2-Dimethyl-1,3-dioxan-5-one (0.52 g,
4.02 mmol) in 1 mL of anydrous THF was then added to the reaction
mixture via syringe and the reaction was allowed to warm to room
temperature with stirring. Additional THF (1 mL) was added after 30
min to help dilute the resulting precipitate and the resulting
mixture was stirred for an additional 30 min., was transferred to a
separatory funnel containing EtOAc (10 mL) and water (10 mL). The
organic phase was collected, washed with brine, and the combined
aqueous extracts were washed with ethyl acetate (2.times.). The
combined organic extracts were dried over MgSO.sub.4, filtered, and
concentrated to provide a dark red crude oil that was purified
using flash chromatography on a silica gel column (300.times.25 mm)
and eluting with 9:1 hexanes-EtOAc to provide a pale yellow solid
intermediate. Yield: 0.57 g (57%); R.sub.f 0.24 (9:1
hexanes-EtOAc); UV .lamda..sub.max 225, 320 nm. .sup.1H NMR
(CDCl.sub.3) .delta. 8.19 (2H, d, J=8.4 Hz), 7.24 (2H, d, J=8.4
Hz), 6.33 (1H, s), 4.62 (2H, s), 4.42 (2H, s), 1.45 (6H, s).
.sup.13C NMR (CDCl.sub.3) .delta. 146.6, 142.7, 141.3, 129.4,
123.9, 121.1, 99.9, 64.4, 60.8, 24.1.
The pale yellow solid intermediate (0.25 g, 1.0 mmol) was diluted
using THF (20 mL), the resulting mixture was treated with 1 N HCl
(10 mL) and allowed to stir for 5 min. To the reaction mixture was
added diethyl ether (150 mL) and water and the resulting mixture
was transferred to a separatory funnel. The organic layer was dried
(MgSO.sub.4), filtered and concentrated to give an oil. The
resulting diol was then taken up in THF-methanol (1:1, 4 mL each,
0.3 M) followed by the addition of Raney Nickel (100 .mu.L, 100
.mu.L/mmol nitro-group, 50% slurry in water) and hydrazine (74
.mu.L, 1.5 eq.). Gas evolution occurred while the reaction mixture
was heated to 50-60.degree. C. After 30 min and 1 h, 1.5 eq. of
hydrazine was added each time. The yellow mixture was filtered
through celite and washed with methanol. The filtrated was
concentrated to provide Compound 75 as an oil which later
crystallized to a yellow solid. Yield: 0.14 g (78%); UV .lamda.max
215, 260 nm. 1H NMR (DMSO) .delta. 7.00 (2H, d, J=8.4 Hz), 6.51
(2H, d, J=8.4 Hz), 6.33 (1H, s), 5.20 (2H, bs), 4.64 (2H, bd), 4.04
(2H, s). 13C NMR (DMSO) .delta. 147.2, 138.1, 129.6, 126.1, 124.6,
113.7, 63.6, 57.5.
Example 27
Preparation of Compound 79
##STR00200##
To a mixture of Compound 75 (BHMS, 0.12 g, 0.67 mmol) in
methanol-dichloromethane (1:2, 4.5 mL total) was added Fmoc-Val-Cit
(0.33 g, 0.67 mmol) followed by EEDQ (0.25 g, 1.0 mmol, 1.5 eq.)
and the resulting reaction was allowed to stir for 15 hours under
inert atmosphere. Additional EEDQ (1.5 eq.) and Fmoc-Val-Cit (1.0
eq.) were then added due to the presence of unreacted BHMS and the
resulting reaction was allowed to stir for 2 days and concentrated.
The resulting residue was triturated using ether to provide a tan
solid intermediate. ES-MS m/z 659 [M+H].sup.+, 681 [M+Na].sup.+; UV
.lamda..sub.max 215, 270 nm. 1H NMR (DMSO) .delta. 10.04 (1H, s),
8.10 (1H, d, J=7.2 Hz), 7.87 (2H, d, J=7.6 Hz), 7.72 (2H, t, J=7.6
Hz), 7.55 (2H, d, J=8.4 Hz), 7.37-7.43 (3H, m), 7.30 (2H, t, J=7.2
Hz), 7.24 (2H, d, J=8.4 Hz), 6.47 (1H, s), 5.96 (1H, t, J=5.2 Hz),
5.39 (1H, s), 4.83 (1H, t, J=5.2 Hz), 4.78 (1H, t, J=5.2 Hz), 4.40
(1H, dd, J=5.2, 8.0 Hz), 4.20-4.30 (3H, m), 4.11 (2H, d, J=4.4 Hz),
4.04 (2H, d, J=5.2 Hz), 3.91 (1H, t, J=7.2 Hz), 2.84-3.06 (2H, m),
1.91-2.03 (1H, m), 1.29-1.74 (4H, m), 0.86 (3H, d, J=6.8 Hz), 0.84
(3H, J=6.8 Hz).
The tan solid intermediate was diluted with DMF (10 mL) and the
resulting mixture was treated with diethylamine (5 mL), allowed to
stir for 1 hour and concentrated to provide a tan solid which was
dried under high vacuum for 3 days. The tan solid was triturated
using EtOAc (10 mL) and further precipitated using ether (80 mL) to
provide a crude residue which was filtered through a sintered glass
funnel and dried in vacuo to afford a light tan intermediate. ES-MS
m/z 436 [M+H].sup.+, 458 [M+Na].sup.+; UV .lamda..sub.max 215, 270
nm.
The light tan intermediate was diluted with DMF (10 mL) and treated
with 6-maleimidocaproic acid hydroxysuccinimde ester (0.16 g, 0.53
mmol, 1 eq.). The reaction was allowed to stir for 18 h, additional
diisopropylethylamine (1.0 eq) was added followed by additional
6-maleimidocaproic acid hydroxysuccinimde ester (0.5 eq.). The
resulting reaction was allowed to stir for 4 hours, after which
time, HPLC indicated that the starting material had been consumed.
The reaction mixture was concentrated to provide a crude residue
that was triturated using EtOAc (10 mL) and then further
precipitated using ether (75 mL). The precipitate was and dried
overnight to provide a tan/orange powdered intermediate. Overall
yield: 0.42 g (quant.). ES-MS m/z 629 [M+H].sup.+, 651
[M+Na].sup.+; UV .lamda..sub.max 215, 270 nm.
The tan/orange powdered intermediate (0.40 g, 0.64 mmol) was
partially dissolved in DMF (20 mL) and to the resulting mixture was
added bis(4-nitrophenyl) carbonate (0.98 g, 3.2 mmol, 5.0 eq.) and
diisopropylethylamine (0.45 mL, 2.5 mmol, 4.0 eq.). The resulting
reaction was allowed to stir for about 4 hours, after which time,
HPLC monitoring indicated that no starting material remained and
that the reaction mixture contained 2 products in a 3:2 ratio (the
desired bis-carbonate and the 1,3-dioxan-2-one, respectively). The
reaction mixture was concentrated and the resulting residue was
triturated using EtOAc (10 mL), then further precipitated using
ether (80 mL) in a one-pot manner. The EtOAc-ether mixture was
filtered and the solid was dried to provide Compound 79 as a tan
powder which was used without further purification.
Example 28
Preparation of Compound 80
##STR00201##
Compound 49 (202 mg, 0.22 mmol, 2.0 eq., 80% pure) and Compound 79
(180 mg, 0.11 mmol, 1.0 eq., 60% pure) were suspended in dry DMF (2
mL, 0.1 M) and to the resulting mixture was added HOBt (3 mg, 22
.mu.mol, 0.2 eq.) followed by pyridine (400 .mu.L, 1/4 v/v DMF).
The resulting reaction was allowed to stir for 16 h, diluted with
DMSO (2 mL) and and the resulting mixture was purified using
preparative HPLC (C.sub.18-RP column, 5.mu., 100 .ANG., linear
gradient of MeCN in water 10 to 100% in 40 min followed by 20 min
at 100%, at a flow rate of 50 mL/min) to provide Compound 80 as a
white solid. Yield: 70 mg (18%). MALDI-TOF MS m/z 2138.9
[M+Na].sup.+, 2154.9 [M+K].sup.+.
Example 29
Preparation of Compound 81
##STR00202##
Compound 81 was made using the method described in Example 1 and
substituting Fmoc-(D)-val-(L)-cit-PAB-OH for Compound 19.
Example 30
Preparation of Compound 82
##STR00203##
Compound 82 was made using the method described in Example 1 and
substituting Fmoc-(L)-val-(D)-cit-PAB-OH for Compound 19.
Example 31
Preparation of Compound 83
##STR00204##
Compound 83 was made using the method described in Example 1 and
substituting Fmoc-(D)-val-(D)-cit-PAB-OH for Compound 19.
Example 32
Preparation of Compound 84
##STR00205##
Compound 84 was made using the method described in Example 14 and
substituting Compound 81 for Compound 21.
Example 33
Preparation of Compound 85
##STR00206##
Compound 85 was made using the method described in Example 14 and
substituting Compound 82 for Compound 21.
Example 34
Preparation of Compound 86
##STR00207##
Compound 86 was made using the method described in Example 14 and
substituting Compound 83 for Compound 21.
Example 35
Preparation of Compound 87
##STR00208##
A mixture of 6-Maleimidocaproic acid (1.00 g, 4.52 mmol, 1.0 eq.),
p-aminobenzyl alcohol (1.11 g, 9.04 mmol, 2.0 eq.) and EEDQ (2.24
g, 9.04 mmol, 2.0 eq.) were diluted in dichloromethane (13 mL). The
resulting reaction was stirred about 16 hr., then concentrated and
purified using flash column chromatography in a step gradient
25-100% EtOAc in hexanes to provide a solid intermediate. Yield:
1.38 g (96%); ES-MS m/z 317.22 [M+H].sup.+, 339.13 [M+Na].sup.+; UV
.lamda..sub.max 215, 246 nm.
The solid intermediate (0.85 g, 2.69 mmol, 1.0 eq.) and
bis(4-nitrophenyl)carbonate (2.45 g, 8.06 mmol, 3.0 eq.) were
diluted in DMF (10 mL), and to the resulting mixture was added
diisopropylethylamine (0.94 mL, 5.37 mmol, 2.0 eq.). The resulting
reaction was stirred for about 1 hr, after which time RP-HPLC
indicated that the reaction was complete. The reaction mixture was
concentrated in vacuo, and the resulting crude residue was
triturated using diethyl ether (about 250 mL) to provide a white
solid intermediate upon filtration. Yield: 1.25 g (96%); UV
.lamda..sub.max 215, 252 nm.
The white solid intermediate (259 mg, 0.0538 mmol, 1.0 eq.), MMAE
(464 mg, 0.646 mmol, 1.2 eq.), and HOBt (14.5 mg, 0.108 mmol, 0.2
eq.) were diluted in pyridine/DMF (1:5, 6 mL), and the resulting
reaction was stirred for about 10 h, after which time RP-HPLC
indicated incomplete reaction. The reaction mixture was
concentrated, the resulting crude residue was diluted using DMF (3
mL), and to the resulting mixture was added diisopropylethylamine
(0.469 mL, 0.538 mmol, 1.0 eq.) and the resulting reaction was
allowed to stir for about 16 hr. The reaction mixture was directly
purified using Chromatotron.RTM. (radial thin-layer chromatography)
with a step gradient (0-5% methanol in dichloromethane), to provide
Compound 87 as a white solid. Yield: 217 mg (38%); ES-MS m/z
1082.64 [M+Na].sup.+; UV .lamda..sub.max 215, 248 nm.
Example 36
Preparation of Compound 88
##STR00209##
Fmoc-val-cit (U.S. Pat. No. 6,214,345 to Firestone et al.) was
suspended in dichloromethane (50 mL) and the resulting mixture was
treated with 33% HBr in HOAc (20 mL), which was added via pipette
over about 5 minutes. After stirring for about 10 minutes, the
reaction mixture was shown to be complete using HPLC. The reaction
mixture was diluted with ice (about 500 mL) and saturated aqueous
sodium bicarbonate was slowly added while stirring until gas
evolution ceased. The resulting gelatinous mass was filtered and
washed with distilled water to provide a solid which was dried
under high vacuum in the presence of P.sub.2O.sub.5 for 24 h. The
resulting tan powdered intermediate (Fmoc-val-cit-PAB-Br) was about
70% pure by HPLC and was used without further purification.
The tan powdered intermediate (30 mg, 40.6 .mu.mol) and Compound 53
(34 mg, 40.6 .mu.mol) were dissolved in DMF (1 mL), and to the
resulting mixture was added diisopropylethylamine (21 .mu.L, 0.12
mmol, 3.0 eq.). The resulting reaction was allowed to stir for 6 h,
diluted with DMSO (1 mL) and immediately purified using
preparative-HPLC (C.sub.12-RP column, 5.mu., 100 .ANG., linear
gradient of MeCN in water (containing 0.1% formic acid) 10 to 100%
in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min),
to provide as a slight tan powdered intermediate. Yield: 5 mg (8%);
ES-MS m/z 1420 [M+H].sup.+, 1443 [M+Na].sup.+; UV .lamda..sub.max
205, 258 nm.
The slight tan powdered intermediate (4 mg, 9.51 .mu.mol) was
diluted using DMF (1 mL) and the resulting mixture was treated with
diethylamine (0.5 mL). The resulting reaction was complete in 1 h
according to HPLC. The reaction mixture was concentrated to provide
an oily solid residue which was triturated with ether (3.times.) to
provide a crude residue. The crude residue was diluted with DMF (1
mL) and to the resulting mixture was added 6-maleimidocaproic acid
hydroxysuccinimide ester (3 mg, 9.5 .mu.mol). The resulting
reaction was allowed to stir at room temperature for about 16 h.
The reaction mixture was directly purified using preparative-HPLC
(C.sub.12-RP column, 5.mu., 100 .ANG., linear gradient of MeCN in
water (containing 0.1% formic acid) 10 to 100% in 40 min followed
by 20 min at 100%, at a flow rate of 25 mL/min) to provide Compound
88 as a slight tan solid. Yield: 3.9 mg (quant); ES-MS m/z 1391
[M+H].sup.+; UV .lamda..sub.max 205, 250 nm.
Example 37
Preparation of Compound 89
##STR00210##
Preparation of Compound 89A
##STR00211##
Compound 89A was prepared using the method described in Example 9
and substituting tripeptide Compound 43 for tripeptide Compound 42,
intermediate
Preparation of Compound 89
Compound 89a (0.13 g, 0.15 .mu.mol, 1.0 mmol), Compound 21 (0.12 g,
0.17 mmol, 1.1 eq.), and HOBt (4 mg, 31 .mu.mol, 0.2 eq.) were
suspended in DMF/pyridine (2 mL/0.5 mL, respectively). The
resulting reaction was allowed to stir for about 4 h, then
diisopropylethylamine (27 .mu.L, 0.15 mmol, 1.0 eq.) was added and
the resulting reaction was allowed to stirred for about 54 h and
concentrated in vacuo. The resulting crude oil was diluted with
DMSO and purified using preparative-HPLC (C.sub.12-RP column,
5.mu., 100 .ANG., linear gradient of MeCN in water (containing 0.1%
TFA) 10 to 100% in 40 min followed by 20 min at 100%, at a flow
rate of 25 mL/min) to provide to a yellow oil that was taken up in
a minimum amount of dichloromethane and precipitated with excess
ether to afford Compound 89 as a tan powder. Yield: 0.15 mg (68%).
ES-MS m/z 1449.14 [M+H].sup.+; UV .lamda..sub.max 215, 258 nm.
Example 38
Preparation of Compound 90
##STR00212##
1,4-Phenylenediamine dihydrochloride (3.06 g, 17 mmoles) and
di-t-butyl dicarbonate (3.69 g, 17 mmoles) were diluted with 30 mL
of dichloromethane. To the resulting mixture was added
diisopropylethylamine (8.83 ml, 50.7 mmoles, 3.0 eq.) and the
resulting reaction was allowed to stirred for 1 hr. The reaction
mixture was transferred to a seperatory funnel and the organic
phase was washed water (3.times.10 ml). The organic layer was
stored at 4.degree. C. for about 15 h and and crystalization of the
product occurred. The crystals were collected by filtration and
washed with cold dichloromethane to provide Compound 90 as a
crystalline solid. (1.2 g, 34%). UV .lamda..sub.max 215, 250 nm.
.sup.1H NMR (DMSO) .delta. 8.78 (1H, bs), 7.04 (2H, bd, J=7.2 Hz),
6.43 (2H, d, J=7.2 Hz), 4.72 (2H, s), 1.41 (9H, s).
Example 39
Preparation of Compound 91
##STR00213##
A solution of cAC10 (10 mg/mL in 25 mM sodium citrate, 250 mM
sodium chloride, 0.02% Tween 80, pH 6.5) was adjusted to pH 7.5 by
addition of 0.3 M sodium phosphate, dibasic. To this pH-adjusted
cAC10 solution, EDTA was added to a final concentration of 5 mM.
The cAC10 solution was then pre-heated to 37.degree. C. by
incubation in a temperature-controlled oven. After the temperature
of the cAC10 solution has equilibrated to 37.degree. C., DTT (from
a stock solution of 10 mM) was added to achieve a final DTT-to-cAC
10 molar ratio of about 3.0 in the reduction reaction (a molecular
weight of 148,500 Da was used for cAC10). The reduction reaction
was then allowed to proceed for 2 hours at 37.degree. C.
At the end of the incubation, the reduction reaction was cooled to
an internal temperature of 2 to 8.degree. C. in an ice-water bath.
The temperature of the solution was kept at 2 to 8.degree. C.
throughout the remaining conjugation steps. The chilled reduction
reaction was subjected to constant-volume diafiltration to remove
excess DTT using a 30 kDa membrane and the buffer was exchanged
into phosphate buffered saline, pH 7.4 (PBS). Following
diafiltration, the concentration of free thiol in the reduced and
diafiltered cAC10 was determined using General Procedure M.
Conjugation is then carried out by addition of a 15% molar excess
of Compound 58 (from a stock solution of 13 mg/mL in DMSO) relative
to the total thiols determined using General Procedure M.
Additional DMSO was added to the conjugation reaction to achieve a
final DMSO concentration of 15% (v/v). The conjugation reaction was
allowed to proceed for a total of 30 min.
At the end of the conjugation reaction, any unreacted excess
Drug-Linker compound was quenched by addition of excess Cysteine
(2.times. molar excess relative to the total thiols determined
using General Procedure M, performed on the reduced and diafiltered
cAC10 to produce the quenched reaction mixture. The quenched
reaction mixture is then purified free of small-molecule
contaminants via constant-volume diafiltration using a 30 kDa
membrane and the buffer was exchanged into PBS, pH 7.4. After
diafiltration, the conjugate was sterile-filtered using a 0.22
micron filter to provide Compound 91 in a clear, colorless
solution.
Example 40
Preparation of Compound 92
##STR00214##
Compound 92 was prepared using the method described in Example 39
using an amount of DTT (from a stock solution of 10 mM) which
provides a final DTT-to-cAC10 molar ratio of about 1.5 in the
reduction reaction.
In Vitro Cytotoxicity Experiments
The cell lines used were H3396 human breast carcinoma (cBR96
antigen positive, cAC10 antigen negative), HCT-116 human colorectal
carcinoma (cBR96 and cAC10 antigen negative), and Karpas human
anaplastic large cell lymphoma (ALCL) (cBR96 antigen negative,
cAC10 antigen positive). These cell lines are available from ATCC.
CD30-positive Hodgkin's Disease (HD) cell line L540 and the ALCL
cell line Karpas 299 were obtained from the Deutsche Sammlung von
Mikroorganism and Zellkulturen GmbH (Braunschweig, Germany).
L540cy, a derivative of the HD line L540 adapted to xenograft
growth, was provided by Dr. Phil Thorpe (U of Texas Southwestern
Medical Center, Dallas, Tex.). Cell lines were grown in RPMI-1640
media (Life Technologies Inc., Gaithersburg, Md.) supplemented with
10% fetal bovine serum.H3396 cells in RPMI containing 10% fetal
bovine serum (referred to as medium) were plated in 96-well plates
at approximately 3,000-10,000 cells/well and allowed to adhere
overnight. The non-adherent Karpas cell line was plated out at
approximately 10,000 cells/well at the initiation of the assay.
Various concentrations of illustrative Compounds of the Invention
in medium were added in triplicate, and after the times indicated
IN FIGS. 1-7, the medium was removed, and the cells were washed
with fresh medium three times. After a 96 hour incubation period at
37.degree. C., Alamar Blue was added and cell viability was
determined 4 hours later as described by Ahmed S A, Gogal R M Jr,
Walsh J E., J. Immunol. Methods, 170, 211-224, 1994.
C.B.-17 SCID (Harlan, Indianapolis, Ind.) mice were used for in
vivo experiments
Example 41
In Vitro Cytotoxicity Data
The cytotoxic effects of Compound 49 and Compound 53 on H3396 human
breast carcinoma cells are shown in FIG. 1. The data show that
after exposure for 1 hour, Compound 53 is more cytotoxic than
Compound 49 at concentrations of up to 0.01 mM. The compounds show
substantially equal cytotoxicity at concentrations between 0.01 mM
and 1.0 mM.
Example 42
In Vitro Cytotoxicity Data
FIG. 2 shows the cytotoxic effects of Compounds 64, 65, 68 and 69
on H3396 human breast carcinoma cells (cBR96 antigen positive,
cAC10 antigen negative). The data show that the Compounds 64 and 68
demonstrate similar and significant cytotoxicity, while Compounds
65 and 69 are less efficacious, but nevertheless cytotoxic against
H3396 cells in this particular assay.
Example 43
In Vitro Cytotoxicity Data
FIG. 3 shows the cytotoxic effects of Compounds 64, 65, 68 and 69
on HCT-116 human colorectal carcinoma cells (cBR96 antigen
negative, cAC10 antigen negative). The data illustrate that none of
Compounds 64, 65, 68 and 69 is cytotoxic toward the antigen
negative HCT-116 cells in this assay.
Example 44
In Vitro Cytotoxicity Data
FIG. 4 illustrates the cytotoxicity of Compounds 66 and 68 on H3396
human breast carcinoma cells (cBR96 antigen positive). The data
show that both Compounds are highly cytotoxic at concentrations
above 0.1 mM and that Compound 68 demonstrates greater cytotoxicity
than Compound 66 at concentrations between 0.01 mg/mL and 0.4
mg/mL.
Example 45
In Vitro Cytotoxicity Data
FIG. 5 illustrates the cytotoxicity of Compounds 66, 68 and 69 on
Karpas human anaplastic large cell lymphoma (cBR96 antigen
negative, cAC10 antigen positive). The data show that Compound 69
was more cytotoxic toward Karpas cells than compared to Compounds
68 and 66 in this assay. Compound 69 demonstrated significant
cytotoxicity at concentrations above 0.001 mM, while Compound 66
and Compound 68 were not cytotoxic at concentrations below 1.0
mg/mL.
Example 46
In Vitro Cytotoxicity Data
FIG. 6 illustrates the cytotoxicity of Compound 66 and 67 at 2 h
and 96 h on H3396 human breast carcinoma cells (cBR96 antigen
positive, cAC10 antigen negative). The data show that Compound 66
is highly cytotoxic at concentrations above 100 mg/mL at short-term
exposure (2 h) mg/mL, and at concentrations above 100 mg/mL over
long-term exposure (96 h). Compound 67 did not demonstrate
cytotoxicity against H3396 cells in this assay at concentrations up
to 1000 mg/mL.
General Procedure S: In Vivo Testing of Selected
Drug-Linker-Antibody Conjugates. For the L2987 human adenocarcinoma
cell line, Athymic nude mice (8-10 weeks old) were implanted with
xenograft tumors or tumor cells. For the Karpas human anaplastic
large cell lymphoma model, CB-17 SCID mice were implanted
subcutaneously with 5.times.10.sup.6 cells. In both tumor models,
therapy was initiated once the tumors reached an average volume of
100 mm.sup.3. Groups of mice were injected with one of Compounds
66-69 in phosphate buffered saline intravenously every fours days
for a total of 6 injections for L2987 animals and 2 injections for
Karpas animals. Tumor volume was computed using the formula: 0.5
(longest dimension.times.perpendicular dimension.sup.2). Mice were
removed from the study when their tumors were approximately 1000
mm.sup.3, at which point the average tumor sizes from the
particular group were no longer plotted.
Example 47
In Vivo Therapeutic Efficacy on 12987 Tumors
FIG. 7 shows the therapeutic effects of Compounds 66-69 on L2987
human lung adenocarcinoma xenograft tumors (cBR96 antigen positive,
cAC10 antigen negative) implanted in athymic nude mice. General
Procedure S was followed using subcutaneous
L2987 human lung tumors (from in vivo passaging). Mice were
administered by injection with one of Compounds 66, 67, 68 or 69 at
four day intervals for a total of 6 injections. The first injection
was given at 15 days post tumor-implant. The data illustrate that
administration of Compound 66 and Compound 68 markedly reduced
tumor volume and no additional growth was noted in treated mice for
at approximately 25 days after the last injection. Compound 67 and
Compound 69 were less efficacious but nevertheless inhibited tumor
cell multiplication in the treated mice. Testing was stopped in
animals receiving Compounds 67 and 69 when tumor volume exceeded
1000 mm.sup.3.
Example 48
In Vivo Therapeutic Efficacy on Karpas Tumors
FIG. 8 shows the therapeutic effects of compounds 66-69 on Karpas
human anaplastic large cell lymphoma xenograft tumors (cAC10
antigen positive, cBR96 antigen negative) implanted in nude mice.
General Procedure S was followed using Karpas human anaplastic
large cell lymphoma model, CB-17 SCID mice were implanted
subcutaneously with 5.times.10.sup.6 cells. Mice were dosed
intravenously with one of Compounds 66, 67, 68 or 69 at four day
intervals for a total of 2 injections starting on day 8. The data
illustrate that Compounds 67 and 69 induced complete regressions,
and that the tumors progressed in animals that received
substantially equivalent amounts of Compounds 66 and 68.
Example 49
Determination of Cytotoxicity of Selected Compounds in CD30- and
CD30+ Cells
Following their physical characterization, the in vitro
cytotoxicity of Compounds 67, 91 and 92 was evaluated in CD30.sup.+
Karpas 299 and CD30.sup.- Raji cells using the Alamar Blue assay as
described above. The percent viable cells was plotted versus
concentration for each molecule to determine the IC.sub.50 (defined
as the mAb concentration that gave 50% cell kill).
Compound 67 demonstrated activity against Karpas 299 cells with an
IC.sub.50 of 4 ng/mL. The IC.sub.50 was inversely proportional to
drug loading as it increased from 4 ng/mL for Compound 67 to 7
ng/mL for Compound 91, to 40 ng/mL for Compound 92. Selectivity of
the tested compounds was evaluated using the antigen-negative Raji
cell line which were insensitive to all cAC10-containing Compounds
with IC.sub.50 values >1000 ng/ml for Compounds 67, 91 and
92.
Example 50
Cytotoxicity of Selected Compounds in Xenograft Models of HD and
ALCL
Cytotoxicity of Compounds 67, 91 and 92 was evaluated in
subcutaneous Karpas 299 human anaplastic large cell lymphoma and
L540cy Hodgkin's Disease xenograft models in C.B.-17 SCID mice.
Evaluations were initiated when tumor volumes averaged 50-100
mm.sup.3. Cohorts of Karpas-299 bearing mice were injected q4dx4
with Compound 92, Compound 91, or Compound 67 at either 0.25 mg/kg
or 0.5 mg/kg. None of the animals treated at 0.25 mg/kg had a
regression, although there was a delay in tumor growth compared to
untreated controls for the animals treated with Compound 91 and
Compound 67. Treatment of Karpas tumors with Compound 91 and
Compound 67 at 0.5 mg/kg given q4dx4 achieved 5/5 complete
regressions and 4/5 complete regressions, respectively. A delay in
tumor growth compared to untreated animals was observed for
Compound 92 at 0.5 mg/kg given q4dx4, but no complete regressions
were obtained.
Efficacy was also tested in a subcutaneous Karpas model with
selected compounds administered as a single dose. Compound 91 and
Compound 67 were injected at single doses of 0.25, 0.5 and 2.0
mg/kg. At the dose of 0.25 mg/kg there was no antitumor activity in
either group and mean tumor volume did not deviate from the
untreated controls. A delay in the tumor growth was demonstrated by
both molecules at 0.5 mg/kg, but no complete regressions were
obtained. Treating the mice with Compound 91 and Compound 67 at 2
mg/kg achieved 100% complete regressions in both groups.
Compound 91 and Compound 67 were also compared in mice bearing
subcutaneous L540cy human HD tumors treated q4dx4 with Compound 91
and Compound 67 at 1 and 3 mg/kg. At 1 mg/kg, mice treated with
Compound 91 and Compound 67 had significant delays in tumor growth
compared to the untreated animals. Complete regressions were
observed in mice administered with both Compound 91 and Compound 67
at 3 mg/kg.
The present invention is not to be limited in scope by the specific
embodiments disclosed in the examples which are intended as
illustrations of a few aspects of the invention and any embodiments
that are functionally equivalent are within the scope of this
invention. Indeed, various modifications of the invention in
addition to those shown and described herein will become apparent
to those skilled in the are and are intended to fall within the
scope of the appended claims.
A number of references have been cited, the entire disclosures of
which are incorporated herein by reference.
* * * * *